Spies in Disguise does not include any discernible LGBTQ+ characters or themes. The narrative focuses on a super spy and a scientist, and their evolving partnership, without touching upon queer identity in any capacity.

Spies in Disguise does not feature any identifiable transsexual characters or themes. The story centers on an animated spy adventure, and the topic of transsexual identity is not present within its narrative or character arcs.

The film features several competent female characters, including Internal Affairs agents Wendy Beckett, Joy Jenkins, and Marcy Kappel. While they participate in action sequences involving chases and the use of gadgets and firearms, there are no scenes where a female character defeats one or more male opponents in direct physical combat, such as hand-to-hand or melee weapon fights.

The film "Spies in Disguise" is an original animated story, loosely based on a short film. Its characters are either new creations or maintain their original gender from the short film, with no instances of established characters being portrayed as a different gender.

Spies in Disguise is an original animated film based on a short. Its characters were created for this production, meaning there is no prior established canon or historical record from which their race could have been swapped.