Background COVID-19 disrupted in-person clinical research activities. In response, the Adolescent Medicine Trials Network (ATN) CARES study instituted mail-based testing for human immunodeficiency virus (HIV), sexually transmitted infections (STIs), and substance use screening. We assessed the acceptability and feasibility of the ATN CARES mail-based testing program. Methods From September 2020- December 2020, trained study staff sent myLAB Box mail-based test kits and written instructions to gay, bisexual, and transgender study participants between the ages of 16-26 years. The test kits included a dried blood spot collection card for HIV (4th generation antigen/ antibody EIA) and syphilis (TP-EIA) testing, swabs and a urine collection kit for the detection of extragenital and genital Chlamydia trachomatis and Neisseria gonorrhoeae infection, and a urine collection kit for the screening of six substances. Staff tracked shipment status, and when delivery was identified, contacted participants to assist them with selfcollection and test kit return. Results Of the 65 youth who participated to date, 49.2% (32/ 65) returned their kit and completed at least one test. Those 32 participants had a median age of 23.5 years (IQR 22-25). The median number of days from test kit ordered to results was 18.5 days (IQR 15-23.5);median number of days from collection to results was 10 days (IQR 8.5-12). MyLAB Box reported a total of 219 test Results 30 HIV, 22 syphilis, 56 chlamydia, 56 gonorrhea, and 55 drug tests processed. Of the 219 tests, 7.3% (16) were rejected due to specimen inadequacy (6 dried blood spot cards, 2 anal swabs, 8 urine specimens). Conclusions Mail-based testing is a promising way to monitor a variety of biological study outcomes among adolescent study participants. Future studies should further investigate how to strengthen utilization and fidelity of mail-based testing.