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Introduction: To ensure the health and safety of persons taking pre-exposure prophylaxis to prevent HIV infection, the 2017 Centers for Disease Control and Prevention guidelines recommended initial and follow-up laboratory testing. We assessed the trends in adherence rates to recommended laboratory testing among pre-exposure prophylaxis users and identified factors associated with HIV testing among pre-exposure prophylaxis users from 2016 to 2020 and also examined rate changes during the COVID-19 pandemic in 2020. Methods: We conducted a retrospective cohort study assessing the rates and trends of recommended laboratory testing among commercially insured pre-exposure prophylaxis users from 2016 to 2020, using the MarketScan database. We examined the proportion of pre-exposure prophylaxis users adhering to the following initial and follow-up laboratory testing: (1) HIV, creatinine clearance, hepatitis B virus, hepatitis C virus, and sexually transmitted infections (chlamydia/gonorrhea and syphilis) within 7 days before pre-exposure prophylaxis initiation; (2) HIV 90 days after initiation; and (3) HIV, creatinine clearance, and sexually transmitted infections 180 days after pre-exposure prophylaxis initiation. We used general linear models to examine trends and multivariable logistic regression to identify predictors of ≥1 HIV test within 180 days after index pre-exposure prophylaxis. Results: We identified 19,581 new pre-exposure prophylaxis users. Most were male (96%) and aged 18–34 years (55%). Adherence rates to recommended testing increased from 2016 through 2019 (e.g., 9.0%–13.6% for all initial screening tests 7 days before initiation, 42.1%–44.6% for HIV testing 90 days after initiation, 33.8%–40.6% for all follow-up tests within 180 days after initiation), but all rates decreased during the COVID-19 pandemic (12.4%, 33.6%, and 31.6%, respectively). Younger age (aged 13–17 years: AOR=0.44, 95% CI=0.28, 0.71) and ages 18–34 years (AOR=0.80, 95% CI=0.74, 0.86) were associated with a significantly lower likelihood of getting an HIV test within 180 days after initiation than ages 35–44 years, and female sex (AOR=0.64, 95% CI=0.55, 0.74) were associated with a significantly lower likelihood than male sex. Pre-exposure prophylaxis users with a history of sexually transmitted infections had a higher likelihood (AOR=1.27, 95% CI=1.16, 1.40) of getting tested than those without. Conclusions: Initial screening and follow-up testing rates were lower than those recommended by the Centers for Disease Control and Prevention. Public health efforts are needed to ensure that patients have access to needed laboratory testing during pandemics or natural disasters and to educate patients and clinicians about the importance of screening and monitoring tests to ensure the safety and health of pre-exposure prophylaxis users.