BACKGROUND: HIV pre-exposure prophylaxis (PrEP) is highly effective for prevention of HIV acquisition, but requires HIV testing at regular intervals. Female sex workers (FSWs) are a priority population for HIV prevention interventions in many settings, but face barriers to accessing healthcare. Here, we assessed the acceptability of HIV self-testing for regular HIV testing during PrEP implementation among FSWs participating in a randomized controlled trial of HIV self-testing delivery models. METHODS: We used data from two HIV self-testing randomized controlled trials with identical protocols in Zambia and in Uganda. From September-October 2016, participants were randomized in groups to: (1) direct delivery of an HIV self-test, (2) delivery of a coupon, exchangeable for an HIV self-test at nearby health clinics, or (3) standard HIV testing services. Participants completed assessments at baseline and 4 weeks. Participants reporting their last HIV test was negative were asked about their interest in various PrEP modalities and their HIV testing preferences. We used mixed effects logistic regression models to measure differences in outcomes across randomization arms at four weeks. RESULTS: At 4 weeks, 633 participants in Zambia and 749 participants in Uganda reported testing negative at their last HIV test. The majority of participants in both studies were "very interested" in daily oral PrEP (91% Zambia; 66% Uganda) and preferred HIV self-testing to standard testing services while on PrEP (87% Zambia; 82% Uganda). Participants in the HIV self-testing intervention arms more often reported preference for HIV self-testing compared to standard testing services to support PrEP in both Zambia (P = 0.002) and Uganda (P < 0.001). CONCLUSION: PrEP implementation programs for FSW could consider inclusion of HIV self-testing to reduce the clinic-based HIV testing burden. TRIAL REGISTRATION: ClinicalTrials.gov NCT02827240 and NCT02846402 .