We aimed to determine whether density gradient centrifugation and magnetic-activated cell sorting (DGC-MACS) could select viable spermatozoa, with lower levels of DNA fragmentation, from an immotile population. Analysis involved sixteen patients, each with a sperm count ≥107/mL. All samples were immotile despite exhibiting a live population >40%. Spermatozoa were prepared using DGC-MACS and selected spermatozoa evaluated for membrane and DNA integrity using the hypo-osmotic swelling (HOS) test, vital staining and the TUNEL test. The mean proportion of spermatozoa with an intact membrane in control, DGC and DGC-MACS populations, was 52.5 ± 12.21%, 69.38 ± 7.87% and 81.81 ± 5.29%. The mean proportion of live spermatozoa in control, DGC and DGC-MACS populations, was 65.88 ± 12.77%, 77.25 ± 7.39% and 85.81 ± 5.2%. DGC-MACS reduced the within-sample discrepancy between HOS test and vital stain results from 13.18% to 4.12%. The mean proportion of spermatozoa exhibiting DNA damage in control, DGC and DGC-MACS populations, was 9.56 ± 3.39%, 5.25 ± 1.61% and 2.75 ± 1.13%. Finally, analysis showed that 71.23% of the DNA-fragmented spermatozoa in unprocessed samples were removed following DGC-MACS and that the addition of MACS to an existing DGC protocol reduced fragmented spermatozoa by a further 26.15% compared to DGC alone. Consequently, DGC-MACS is a clinically viable method able to select viable spermatozoa with lower levels of DNA fragmentation from an immotile population.