Sperm chromatin integrity may affect the outcomes of assisted reproductive technology (ART). Developing a clinically reliable strategy to enrich sperm samples with high chromatin quality spermatozoa prior to sperm banking or use in ART would thus be advantageous. The objectives of this study were to: (i) assess the sperm chromatin quality in men with different categories of semen parameters; and (ii) evaluate the extents of Annexin-V magnetic-activated cell sorting (MACS) technology coupled with differential density gradient centrifugation (DGC) in improving sperm chromatin quality. Three categories of men from couples attending a university-based fertility clinic were recruited based on their semen parameters: normozoospermic (n = 13), asthenoteratozoospermic (n = 17) and teratozoospermic (n = 12). For each patient, spermatozoa in semen samples were processed first by DGC to enrich the motility and further by MACS to remove spermatozoa showing apoptotic features. The yield and enrichment of sperm quality was evaluated at each step with conventional semen parameters in conjunction with a combination of five complementary assays, to assess sperm maturity, chromatin structure, compaction and DNA integrity (Hyaluronic Binding Assay, SCSA, chromomycine A3 staining and TUNEL and COMET assays). Our results demonstrated that, compared with normozoospermic samples, raw asthenoteratozoospermic and teratozoospermic samples had a higher proportion of spermatozoa containing DNA breaks, but only asthenoteratozoospermic exhibited altered chromatin structure and decreased binding to hyaluronic acid. Interestingly, the DGC appeared to select for more mature spermatozoa with high DNA compaction. More importantly, in all categories of semen samples, Annexin-V MACS allows enrichment of spermatozoa with good chromatin quality as measured by the TUNEL and SCSA. Because effective treatment modalities to improve sperm DNA damage are limited, our results suggest a potential clinical value of MACS as a mean to enhance sperm quality that may improve assisted reproductive outcomes.