Acetanilide can be produced by reacting acetic anhydride with aniline:[7] The preparation used to be a traditional experiment in introductory organic chemistry lab classes,[8] but it has now been widely replaced by the preparation of either paracetamol or aspirin, both of which teach the same practical techniques (especially recrystallization of the product) but which avoid the use of aniline, a suspected carcinogen.

Acetanilide is used as an inhibitor of hydrogen peroxide decomposition and is used to stabilize cellulose ester varnishes.

[10] In the 19th century acetanilide was one of a large number of compounds used as experimental photographic developers.

Acetanilide was the first aniline derivative found to possess analgesic as well as antipyretic properties, and was quickly introduced into medical practice under the names of Antifebrin by A. Cahn and P. Hepp in 1886.

[13] After several conflicting results over the ensuing fifty years, it was established in 1948 that acetanilide was mostly metabolized to paracetamol (acetaminophen) in the human body, and that it was this metabolite that was responsible for the analgesic and antipyretic properties.