Abstract. Health care workers’ attitudes toward people living with HIV/AIDS (PLWHA) can play an important role in the quality of life of PLWHA as well as in public health efforts in HIV prevention and treatment. The main aim of this study was to develop and evaluate the psychometric properties of an instrument to measure health care workers’ attitudes toward PLWHA using bifactor and IRT analyses. We applied bifactor and Item Response Theory (IRT) analyses to questionnaire development, evaluation, and refinement. A sample of 836 Italian health care workers was recruited in 2013, including medical practitioners, nurses, health care assistants, social workers, and psychologists. We tested a hierarchical item response model (bifactor model) with model fit comparisons with one-dimensional and five-dimensional models. The bifactor model was found to provide the best fit to the observed data. The results were used to construct a 15-item short form. The test information function showed that the scale was slightly more precise at higher levels of negative attitudes. Finally, the differential item functioning (DIF) analysis revealed that the items did not function differently across gender or professional groups. The instrument was found to have good criterion validity with regard to discriminatory practices toward HIV-infected patients.