Sensitive and specific detection of anti- antibodies in standard enzyme-linked immunosorbent assays (ELISAs) is compromised by cross-reactivity and poor sensitivity of classical antigens. Previously, we discovered 48 strongly reactive peptide antigens of -specific B-cell epitopes from 21 immunodominant proteins. By comprehensive individual testing of 11 top-ranked peptide antigens, we found very high sensitivity and specificity for detection of anti- antibodies in chemiluminescent ELISAs. The current study established a labor-saving colorimetric ELISA by using a mixture of 12 strongly reactive peptide antigens (Ctr Mix1) in a single well/serum rather than assaying reactivity to each individual peptide. For performance evaluation, we used a simulated population of 212 anti- antibody-positive and -negative sera from 125 women with NAAT-confirmed active infection and from 87 healthy women at low risk for infection. In comparison to a composite reference standard (CRS) for anti- antibody status, the Ctr Mix1 IgG ELISA achieved 93.9% sensitivity, significantly superior to the 49% to 79% sensitivities of four commercial anti- IgG ELISAs, and 98% specificity of all tested assays. Compared to the labor-intensive individual peptide testing, this mixed peptide ELISA retained high specificity with only marginal, ∼5% sensitivity loss. By ROC-AUC, likelihood ratio, and predictive value analyses, the Ctr Mix1 ELISA performed satisfactorily at 10% to 75% prevalence range of anti- antibodies but significantly better than commercial ELISAs. Thus, the labor-saving mixed peptide colorimetric ELISA format provides simultaneously high specificity and sensitivity for detection of anti- antibodies. For detection of anti- antibodies by serological assays, use of classical chlamydial antigens results in high cross-reactivity and poor sensitivity. Previously, we discovered 48 strongly reactive peptide antigens of -specific B-cell epitopes from 21 immunodominant proteins, and individual testing and combined scoring of 5 to 11 peptide antigens provided highly sensitive and specific detection of anti- antibodies in chemiluminescent ELISAs. To simplify this method, this study established a single-well labor-saving colorimetric ELISA using a mixture of 12 strongly reactive peptide antigens (Ctr Mix1) for detection of anti- antibodies. This Ctr Mix1 ELISA (94% sensitivity and 98% specificity) outperformed 4 commercial ELISAs (49% to 79% sensitivity and 98% specificity). This ELISA can be easily implemented and commercialized, with convenient setup for use in nonspecialized laboratories. Thus, this mixed peptide assay with superior specificity and sensitivity will improve serodiagnosis of infections.