Although there is research interest to assess attitudes on psychedelics, no validated instrument exists for this purpose. We aimed to develop and examine the psychometric properties of the Attitudes on Psychedelics Questionnaire (APQ) in a sample of the Croatian general population. A cross-sectional, web-based survey among the general population was conducted on 1153 participants (62.1% female, 77.7% with a graduate or high school degree, 15.1% health care workers). We assessed participants’ ability to recognize psychedelic substances using a short knowledge test. The APQ consists of 20 items with four sub-scales: Legal Use of Psychedelics, Effects of Psychedelics, Risk Assessment of Psychedelics, and Openness to Psychedelics. This model demonstrated best fit in a confirmatory factor analysis. Total scale reliability was excellent (McDonald’s ω = 0.949, 95% CI = 0.944–0.953). A strong correlation with a similar unvalidated measure (r = 0.885, P < 0.001) demonstrated convergent validity. We observed an association between attitudes and knowledge on psychedelics (r = 0.494, P < 0.001). Younger age, male gender, and lower educational status were associated with higher APQ scores. The APQ is valid, reliable, and could be applied in assessing educational interventions, patients’ treatment outcomes, and the attitudes of different groups of experts. We encourage further validation of the APQ in English.