Background: Burnout syndrome (BS) among healthcare professionals in Peru demands immediate attention. Consequently, there is a need for a validated and standardized instrument to measure and address it effectively. This study aimed to determine the psychometric properties of the Maslach Burnout Inventory (MBI) among healthcare professionals in the Ancash region of Peru. Methods: Using an instrumental design, this study included 303 subjects of both sexes (77.56% women), ranging in age from 22 to 68 years (M = 44.46, SD = 12.25), selected via purposive non-probability sampling. Appropriate content validity, internal structure validity, and item internal consistency were achieved through confirmatory factor analysis, and discriminant validity for the three dimensions was obtained. Evidence of convergent validity was found for the Emotional Exhaustion (EE) and Personal Accomplishment (PA) dimensions, with reliability values (ω > .75). Results: The EE and PA dimensions exhibited acceptable levels of reliability (ω and α > .80). However, the Depersonalization (DP) dimension demonstrated significantly lower reliability (α < .60 and ω < .50). Conclusions: A correlated three-factor model was confirmed, with most items presenting satisfactory factor loadings and inter-item correlations. Nonetheless, convergent validity was not confirmed for the DP dimension.