During the 2015-2016 school year, the Florida Department of Health in Duval County hosted Teen Health Centers (TeenHC) at five high schools of Jacksonville providing HIV/STD screening and pregnancy testing. The purpose of this study was to assess the cost-effectiveness of the TeenHC chlamydia screening program and determine at what student participation level, the program can be cost-effective. We assessed the costs and effectiveness of the chlamydia screening program compared with "no TeenHC". Cost-effectiveness was measured as cost per quality-adjusted life years (QALY) gained. At a program cost of US$61,001 and 3% participation rate, the cost/QALY gained was $124,328 in the base-case analysis and $81,014-$264,271 in 95% of the simulation trials, all greater than the frequently citied $50,000/QALY benchmark. The cost/QALY gained could be <$50,000/QALY if student participation rate was >7%. The TeenHC chlamydia screening has the potential to be cost-effective. Future program efforts should focus on improving student participation.