Droplet digital PCR (ddPCR) is an emulsion PCR process that performs absolute quantitation of nucleic acids. We developed a ddPCR assay for Chlamydia trachomatis infections and found it to be accurate and precise. Using PCR mixtures containing plasmids engineered to include the PCR target sequences, we were able to quantify with a dynamic range between 0.07 and 3,160 targets/μl (r(2) = 0.9927) with >95% confidence. Using 1,509 clinical conjunctival swab samples from a population in which trachoma is endemic in Guinea Bissau, we evaluated the specificity and sensitivity of the quantitative ddPCR assay in diagnosing ocular C. trachomatis infections by comparing the performances of ddPCR and the Roche Amplicor CT/NG test. We defined ddPCR tests as positive when we had ≥95% confidence in a nonzero estimate of target load. The sensitivity of ddPCR against Amplicor was 73.3% (95% confidence interval [CI], 67.9 to 78.7%), and specificity was 99.1% (95% CI, 98.6 to 99.6%). Negative and positive predictive values were 94.6% (95% CI, 93.4 to 95.8%) and 94.5% (95% CI, 91.3 to 97.7%), respectively. Based on Amplicor CT/NG testing, the estimated population prevalence of C. trachomatis ocular infection was ∼17.5%. Receiver-operator curve analysis was used to select critical cutoff values for use in clinical settings in which a balance between higher sensitivity and specificity is required. We concluded that ddPCR is an effective diagnostic technology suitable for both research and clinical use in diagnosing ocular C. trachomatis infections.