To define physiologic levels of reactive oxygen species in infertile men and establish a cutoff value of reactive oxygen species level in neat semen with a high sensitivity and specificity to differentiate infertile men from fertile donors (controls).Reactive oxygen species levels were measured in the neat semen samples (n = 51) from fertile donors and infertile patients (n = 54).Reproductive research laboratory at a tertiary care hospital.Infertile patients from male infertility clinic.Reactive oxygen species measurement in neat semen sample using luminol-based chemiluminescence method, receiver operating characteristic curves.Seminal reactive oxygen species levels, cutoff value, sensitivity and specificity, positive and negative predictive values.The best cutoff value to distinguish between healthy fertile donors and infertile men was 0.0185 × 10 6 counted photons per minute/20 × 10 6 sperm. At this threshold, the specificity was 82% and the sensitivity was 78%. This value can be defined as basal reactive oxygen species level in infertile men.Reactive oxygen species levels in neat semen samples as measured by luminol-based chemiluminescence are a highly specific and sensitive test in the diagnosis of infertility. This test also may help clinicians treat patients with seminal oxidative stress.