Oxidative stress is increasingly recognized as an important contributor to male infertility, yet current methods for measuring reactive oxygen species and free radicals in spermatozoa lack single-cell resolution and often rely on indirect or non-specific detection strategies. Diamond relaxometry, a quantum sensing technique based on nitrogen-vacancy centers in fluorescent nanodiamonds (FNDs), enables direct detection of local free radical production in living cells. Although previously demonstrated in boar spermatozoa, application at the level of individual human sperm cells required further methodological optimization. This study aimed to translate and optimize T1 relaxometry from a pre-clinical boar sperm model to human spermatozoa. A stepwise optimization strategy was applied in boar spermatozoa, systematically evaluating incubation time, temperature and processing media focusing on FNDs colocalization with spermatozoa head, cell metabolic activity, and protocol-induced effects on T1 measurements. The optimized protocol was subsequently translated to human spermatozoa. We showed that incubation time of 1h (vs. 3h), incubation temperature at room temperature (vs. 37°C) and albumin-containing processing medium, yielded a clinically compatible protocol that preserved sperm metabolic activity while ensuring robust FND binding and stable T1 relaxation times. Using the optimized protocol, significant reductions in T1 relaxation time were observed following menadione treatment in both boar (1 mM) and human spermatozoa (10 μM), demonstrating sensitivity to increases in free radical production at the single-cell level (p<0.001 and p=0.043, respectively). In human spermatozoa, FND colocalization reached 36%, enabling successful relaxometry measurements. In conclusion, we successfully optimized and translated diamond relaxometry from boar to human spermatozoa and established a clinically compatible protocol for single-cell free radical detection in human spermatozoa. This work demonstrates the feasibility of quantum sensing in individual human sperm cells and provides a foundation for future studies investigating oxidative stress as a biomarker in male infertility.