Distinguishing human spermatozoa carrying X or Y chromosomes presents a significant analytical challenge because both populations are nearly identical in morphology, physiology, and biochemical composition. The most consistent measurable difference lies in nuclear DNA content, with X-bearing sperm containing approximately three percent more DNA than Y-bearing sperm. Traditional approaches such as density gradient centrifugation and motility-based selection lack sufficient analytical precision and show variable reproducibility.

In contrast, modern bioanalytical technologies—including flow cytometry, Raman-based spectroscopic analysis, microfluidic devices, dielectrophoretic manipulation, nanotechnology-assisted platforms, and artificial intelligence- driven imaging—have substantially improved discrimination sensitivity. This review critically examines these advanced methodologies from an applied bioanalysis perspective, emphasizing their analytical principles, separation efficiency, validation status, and translational feasibility. Ethical and regulatory constraints relevant to human applications are also discussed. At present, flow cytometric sperm sorting remains the most reliable and clinically validated technique, while emerging hybrid platforms show promise for future innovation.