Abstract Introduction Vaccinаtion health literacy (VHL) is considered an extremely important aspect of health literacy. HLS19-VAC questionnaire is a 14-item ‘subjective’ perception-based instrument used for measuring VHL in the M-POHL Health Literacy Survey 2019-2021, which Serbia was not part of. Aim Translation and cultural validation of the HLS19-VAC questionnaire and using the validated Serbian version (HLS19-VAC_RS) for pilot study research. Materials and methods The English version of HLS19-VAC was translated using a systematic approach; three forward and one back-translations were synthesized and compared by an expert committee. Thereafter, cognitive interviews with 11 participants produced a version that was culturally validated by an expert panel. We conducted a cross-sectional pilot study using a HLS19-VAC_RS, on a convenient sample of the general population in Serbia. VHL was assessed by calculating the score for 4 out of 14 questions. Additional data were collected through a general questionnaire with 30 questions. Descriptive and inferential statistics were used. Results Cognitive interviews showed some problems in wording, and cultural adaptation resulted in minor refinements to eight out of 14 items. The instrument was pretested by 11 experts who adjusted it accordingly to produce culturally validated HLS19 -VAC_RS. Of 439 respondents in a pilot study, 15% had problematic VHL, while the levels of sufficient and excellent VHL were nearly the same (38.5% and 35.1%, respectively). There was difficulty in deciding whether to receive the flu vaccine (15.0%) and to find information about recommended vaccines (12.3%). There was a significant statistical difference in responses to VHL and monthly income and alcohol consumption. Most of the respondents contacted pharmacists as primary sources of information about medication (42.7%). Conclusions HLS19 -VAC_RS could be implemented in a local context as culturally appropriate. Further testing on a population level is needed. Key messages • HLS19-VAC_RS Questionnaire is culturally appropriate for assessing vaccination health literacy in Serbia. • Majority of respodents had sufficient or excellent vaccination health literacy.