Background: Psychoactive substance use is the ingestion of substances or the deliberate use of substances for purposes other than those intended. Psychoactive substance use is on the rise among University students of Ethiopians. Objective: To asses prevalence and factors associated with psychoactive substance use among undergraduate students of Hawassa University College of Medical and Health Science, Hawassa, 2022. Methods: Institutional based mixed study method was employed among Hawassa University College of Medicine and Health Science students. The data were collected from Feb14-May6/2022 using online self-administered questionnaire which developed on the Google form for quantitative method and purposive sampling technique was used for qualitative method. A total of 614 Students was selected using a stratified random sampling technique. In depth interviews was conducted with students and key informants. The Quantitative data were exported to SPSS version 25, cleaned and edited before analysis. Logistic regression model was used to determine prediction levels of independent variables to the outcome variables and a variable having P<0.05 was considered as statistically significant variable in all tests. Qualitative data was analyzed manually using thematic analysis and presented in narration. Result: The overall prevalence of current substance is 14.8[11.9, 17.7]. Family member of substance use (AOR (95%CI) = 4.52(2.43, 8.4), relationship status of students (AOR (95%CI) =2.95(1.39, 6.25), acceptance of substance in community (AOR (95%CI) = 2.84(1.35, 5.96), close friend substance use (AOR (95%CI) = 6.4(3.3, 12.39), marital status of students’ family (AOR (95%CI) =3.8(1.45, 9.93), year of study or being intern (AOR (95%CI) =6.8(2.05, 12.56) were found to be significantly associated with students’ psychoactive substance use. Conclusion: According to the finding from this study use of psychoactive substance use among University students in the study area was relatively high. The most utilized substance was alcoholic beverage followed by tobacco products and khat. Year of class , relationship status of students, status of marriage of family, acceptance by community, family member who use substance and close friends who use substance were factors associated with use of psychoactive substances