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The United States’ initiative to End the HIV Epidemic by 2030 includes a primary goal to reduce new HIV infections by 90 percent. One key contributor to this plan is HIV pre-exposure prophylaxis (PrEP). While knowledge and acceptance of PrEP among clinicians is growing, few studies have assessed knowledge and awareness among future healthcare professionals in academic training programs. The present study aimed to assess and compare healthcare trainees’ awareness, knowledge, and familiarity with PrEP prescribing guidelines to better understand and prevent gaps in academic training regarding PrEP. A cross-sectional web-based survey of medical, nurse practitioner, and pharmacy students enrolled at two universities was conducted between October 2017 and January 2018. The study assessed participants’ awareness, knowledge, and familiarity with PrEP prescribing guidelines and willingness to prescribe PrEP and refer to another healthcare provider. The survey was completed by 744 participants (response rate = 36.2%). Overall, PrEP awareness was high though PrEP knowledge was low. There were significant differences among student groups in domains of interest. Pharmacy students had the greatest PrEP knowledge, awareness, and familiarity with prescribing guidelines. However, medical students reported the greatest comfort with performing PrEP-related clinical activities and willingness to refer a candidate to another provider. Study findings enhance our understanding of healthcare professional students’ perspectives of PrEP as a biomedical prevention strategy for HIV. The gaps in students’ knowledge offer opportunities for the development of educational strategies to support HIV prevention among future healthcare professionals.