Director Nate Fox stated that much of the inspiration of the superhero story came from two DC Comics series, DMZ and No Man's Land. Both center on a city after a large disaster. The series also inspired the crafting of the game's grim take on the superhero genre. Fox further stated that the film Batman Begins was an inspiration for the game. However, any correlation to DC Comics' Static Shock was unintentional. Fox considered his own personal involvement in the Seattle WTO riots of 1999 as influencing the reflection of "spending time in a lawless place" within the game.

Grand Theft Auto III was also stated as an influence, in that the team could easily see themselves as superheroes in the open world of Liberty City; similarly, Spider-Man 2 was used as a model to demonstrate what it would be like to move about the city and answer random requests for help alongside regular missions.

In June 2009, game journalist Ben "Yahtzee" Croshaw uploaded video reviews of the games Infamous and Prototype as part of his employer The Escapist's web series Zero Punctuation. In his review of Prototype, he compared both games for having similar premises and being of similar quality (each game released a few weeks apart), but to determine which was the best, he jokingly challenged both games' developers to submit an image of their competitor's protagonists wearing a woman's bra. The developers unexpectedly took the challenge seriously and one-upped it the following week, with Sucker Punch Productions producing a portrait of a gender-swapped Alex Mercer, and Radical Entertainment producing an image of Cole MacGrath as a drag queen in lingerie. Croshaw followed up by reviewing the two images, noting that they had similarities even beyond the prompt, including unicorns and rainbows: