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Dr. Henry Jekyll is a doctor who feels that he is battling between the benevolence and malevolence within himself, thus leading to the struggle with his alter ego Edward Hyde. He spends his life trying to repress evil urges that are not fitting for a man of his stature. Jekyll develops a serum in an attempt to mask this hidden evil. However, in doing so, Jekyll transforms into Hyde, a hideous creature without compassion or remorse. Jekyll has a friendly personality, but as Hyde, he becomes mysterious and violent. As time goes by, Hyde grows in power and eventually manifests whenever Jekyll shows signs of physical or moral weakness, no longer needing the serum to be released.

Stevenson never says exactly what Hyde does, generally saying that it is something of an evil and lustful nature. Thus, in the context of the times, it is abhorrent to Victorian religious morality. Hyde may have been reeling in activities such as engaging with prostitutes or buggery. However, it is Hyde's violent activities that seem to give him the most thrills, driving him to attack and murder Sir Danvers Carew without apparent reason, making him a hunted outlaw throughout England. Carew was a client of Gabriel Utterson, Jekyll's lawyer and friend, who is concerned by Hyde's history of violence and the fact that Jekyll changed his will, leaving everything to Hyde. Dr. Hastie Lanyon, a mutual acquaintance of Jekyll and Utterson, dies of shock after receiving information relating to Jekyll. Before his death, Lanyon gives Utterson a letter to be opened after Jekyll's death or disappearance.

When Jekyll refuses to leave his lab for weeks, Utterson and Jekyll's butler Mr. Poole break into the lab. Inside, they find the body of Hyde wearing Jekyll's clothes and apparently dead from suicide. They find also a letter from Jekyll to Utterson promising to explain the entire mystery. Utterson takes the document home where he first reads Lanyon's letter and then Jekyll's. The first reveals that Lanyon's deterioration and eventual death resulted from seeing Hyde drinking a serum or potion and subsequently turning into Jekyll. The second letter explains that Jekyll, having previously indulged unstated vices (and with it the fear that discovery would lead to his losing his social position), found a way to transform himself and thereby indulge his vices without fear of detection. But Jekyll's transformed personality Hyde was effectively a sociopath — evil, self-indulgent, and utterly uncaring to anyone but himself. Initially, Jekyll was able to control the transformations, but then he became Hyde involuntarily in his sleep.

At this point, Jekyll resolved to cease becoming Hyde. One night however, the urge gripped him too strongly. After the transformation, he immediately rushed out and violently killed Carew. Horrified, Jekyll tried more adamantly to stop the transformations. For a time, he proved successful by engaging in philanthropic work. One day at a park, he considered how good a person he had become as a result of his deeds (in comparison to others), believing himself redeemed. However, before he completed his line of thought, he looked down at his hands and realized that he had suddenly transformed once again into Hyde. This was the first time that an involuntary metamorphosis had happened in waking hours. Far from his laboratory and hunted by the police as a murderer, Hyde needed help to avoid being caught. He wrote to Lanyon (in Jekyll's hand) asking his friend to retrieve the contents of a cabinet in his laboratory and to meet him at midnight at Lanyon's home in Cavendish Square. In Lanyon's presence, Hyde mixed the potion and transformed back to Jekyll - ultimately leading to Lanyon's death. Meanwhile, Jekyll returned to his home only to find himself ever more helpless and trapped as the transformations increased in frequency and necessitated even larger doses of potion in order to reverse them.

Eventually, the stock of ingredients from which Jekyll had been preparing the potion ran low, and subsequent batches prepared by Dr. Jekyll from renewed stocks failed to produce the transformation. Jekyll speculated that the one essential ingredient that made the original potion work (a salt) must have itself been contaminated. After sending Poole to one chemist after another to purchase the salt that was running low only to find it wouldn't work, he assumed that subsequent supplies all lacked the essential ingredient that made the potion successful for his experiments. His ability to change back from Hyde into Jekyll had slowly vanished in consequence. Jekyll wrote that even as he composed his letter, he knew that he would soon become Hyde permanently, having used the last of this salt and he wondered if Hyde would face execution for his crimes or choose to kill himself. Jekyll noted that in either case, the end of his letter marked the end of his life. He ended the letter saying "I bring the life of that unhappy Henry Jekyll to an end". With these words, both the document and the novella come to a close.