This study was aimed to determine the efficiency of synthetic protein-free media in spermatozoa washing, preparationand retention of the activity of washed spermatozoa over short periods in vitro. Normozoospermic semen samples (n =71) were equally apportioned and washed using synthetic protein-free medium (PFM), minimum essential medium + HSA(MEM) or commercial protein-containing medium (CPC). Washed spermatozoa were cultured in vitro using PFM, MEM orCPC media and held for 24 hrs at 4°C, 15°C, 22°C or 37°C. Spermatozoa activity was evaluated at 0 hr, 4 to 7 hrs and24 hrs post-wash. The effects of PFM on spermatozoa motility, vitality, membrane integrity and DNA fragmentation levelwere not significantly different from that of MEM and CPC media at 0 hr, 4 to 7 hrs and 24 hrs post-wash in vitro. SyntheticPFM, MEM and CPC retained spermatozoa activity highest when specimen were held at 22°C and it was significantly higher(p < 0.05) than that at 37°C after 24 hrs incubation in vitro. However, no significant changes (p > 0.05) were notedin spermatozoa DNA fragmentation (SDF) levels when specimen were held at 22°C or 37°C at 4 to 7 hrs and also after24 hrs post-wash in vitro in all media. The use of synthetic PFM as an alternative to the commercial protein-containingmedia in human spermatozoa washing and preparation procedure for an efficient and safer (Assisted ReproductionTechnology) ART outcome. Spermatozoa activity can be successfully retained at room temperature post-wash over shortperiods; spermatozoa may lose viability rapidly if held for long hours at 37°C in a