We report findings from a validation study of the translated and culturally adapted Serbian version of Maslach Burnout Inventory-Human Services Survey (MBI-HSS), for a sample of anesthesiologists working in the tertiary healthcare. The results showed the sufficient overall reliability (Cronbach's α = 0.72) of the scores (items 1-22). The results of Bartlett's test of sphericity (χ(2) = 1983.75, df = 231, p < 0.001) and Kaiser-Meyer-Olkin measure of sampling adequacy (0.866) provided solid justification for factor analysis. In order to increase sensitivity of this questionnaire, we performed unfitted factor analysis model (eigenvalue greater than 1) which enabled us to extract the most suitable factor structure for our study instrument. The exploratory factor analysis model revealed five factors with eigenvalues greater than 1.0, explaining 62.0% of cumulative variance. Velicer's MAP test has supported five-factor model with the smallest average squared correlation of 0,184. This study indicated that Serbian version of the MBI-HSS is a reliable and valid instrument to measure burnout among a population of anesthesiologists. Results confirmed strong psychometric characteristics of the study instrument, with recommendations for interpretation of two new factors that may be unique to the Serbian version of the MBI-HSS.