To compare pain control during vulvar biopsy after either application of 5% lidocaine-prilocaine cream or injection of 1% lidocaine. In a single-site randomized trial, patients who needed vulvar biopsy on a non-hair-bearing surface were recruited from a gynecologic oncology clinic to compare lidocaine-prilocaine cream (placed at least 10 minutes before biopsy) with lidocaine injection (at least 1 minute prior). A sample size of 53 participants in each arm (N=106) was planned. Pain was recorded using a 100 mm visual analog scale at three time points: baseline, after application of anesthesia, and after biopsy. The primary outcome was highest pain score recorded. Secondary outcomes were pain score at biopsy, patient experience, and tolerability and acceptability. Linear regression was used to compare the primary outcome between arms while controlling for baseline vulvar pain. A convenience analysis was performed in March 2019. From October 2018 to March 2019, 38 patients completed informed consent and were randomized. Participants were women with median age of 60 years. Most characteristics between groups were similar. Nineteen were analyzed in the lidocaine-prilocaine group, and 18 were analyzed in the lidocaine injection group. The median highest pain score in the lidocaine-prilocaine group was 20.0 mm vs 56.5 mm in the lidocaine injection group. Controlling for baseline pain, the highest pain score in the lidocaine-prilocaine arm was 25.7 mm lower than in the lidocaine injection arm (95% CI [-45.1 to -6.3]; P<.01). Patients randomized to lidocaine-prilocaine had a significantly better experience than those receiving injected lidocaine (median experience score 2.0 mm vs 17.0 mm; P=.02). Lidocaine-prilocaine cream before vulvar biopsy resulted in a lower maximum pain score and a significantly better patient rating of the biopsy experience when compared with lidocaine injection. Lidocaine-prilocaine cream, alone, is a reasonable option to use for vulvar biopsy. ClinicalTrials.gov, NCT03654417.