Purpose <p>Sperm DNA damage is linked to male infertility and poor reproductive outcome. Single-stranded DNA (ssDNA) damage in sperm is the most common type of damage yet is not specifically targeted by the commonly used DNA damage assays. This study aimed to quantitatively detect sperm ssDNA damage using a novel, direct method.</p> Methods <p>The sperm repair-assisted damage detection (SRADD) assay is a single-molecule technique that uses specific repair enzymes to excise damaged segments and incorporates fluorescently labeled nucleotides, enabling visualization and quantification of the damage. We assessed ssDNA damage in sperm donors following induced damage using varying concentrations of H₂O₂. The assay was evaluated for sensitivity, repeatability, and reproducibility. SRADD results were compared with the direct TUNEL assay and the indirect sperm chromatin dispersion (SCD) assay.</p> Results <p>SRADD demonstrated high inter-slide reproducibility (ICC = 0.95). Sensitivity was confirmed by quantifying induced damage in a dose-dependent manner (0.5, 1, 1.5 mM of H₂O₂), demonstrating mean damage ratios of 1.06, 2.16, and 4.83 relative to control, respectively. Baseline damage levels exhibited strong positive correlation with increased induced damage (<i>r</i> = 0.91, <i>p</i> &lt; 0.001). Analysis of healthy sperm donors (<i>n</i> = 59) revealed that 8.5% of men with normal sperm parameters presented with high ssDNA damage levels. SRADD had a moderate correlation with SCD assay and no correlation with conventional semen parameters and TUNEL assay.</p> Conclusion <p>The SRADD assay quantifies sperm ssDNA with high sensitivity and can process dozens of samples simultaneously, making it valuable for andrology and toxicology research and potentially useful in clinical settings such as sperm banks and male-infertility assessment.</p> <p>Trial registration: (6573–19-SMV).</p>