An improved understanding of behaviors reflecting acute pain in cats is a priority for feline welfare. The aim of this study was to create and validate a comprehensive ethogram of acute pain behaviors in cats that can discriminate painful versus non-painful individuals. An inventory of behaviors (ethogram) with their respective descriptors was created based on a literature review of PubMed, Web of Science and CAB Abstracts databases. The ethogram was divided into ten behavior categories that could be evaluated by duration and/or frequency: position in the cage, exploratory behaviors, activity, posture and body position, affective-emotional states, vocalization, playing (with an object), feeding, post-feeding and facial expressions/features. Thirty-six behaviors were analyzed independently by four veterinarians with postgraduate qualifications in feline medicine and/or behavior as (1) not relevant, (2) somewhat relevant, (3) quite relevant or (4) highly relevant and used for content (I-CVI) and face validity. Items with I-CVI scores > 0.67 were included. Twenty-four behaviors were included in the final ethogram. Thirteen items presented full agreement (i.e., I-CVI = 1): positioned in the back of the cage, no attention to surroundings, feigned sleep, grooming, attention to wound, crouched/hunched, abnormal gait, depressed, difficulty grasping food, head shaking, eye squinting, blepharospasm and lowered head position. Seven descriptors were reworded according to expert suggestions. The final ethogram provides a detailed description of acute pain behaviors in cats after content and face validity and can be applied to the characterization of different acute painful conditions in hospitalized cats.