Aim: To elucidate the concentration of the protein and cholesterol in different fractions of human semen from different infertile categories and comparing them with the fertile group. Materials and methods: The human semen was collected from different infertile categories including oligoasthenospermia, asthenospermia, azoospermia, normospermia, oligospermia and fertile group. Immediately after collection, the semen analysis was done as per WHO standard protocols. After that, the semen was centrifuged to get the different fractions. Four main fractions were obtained, (1) spermatozoa, (2) debris or material that precipitates at 12 K rpm for 10 minutes, (3) prosta- somes which was precipitated at 20K rpm for 120 minutes, (4) seminal plasma. The protein concentration was done by Lowry's method and cholesterol was estimated by diagnostic kit. Results: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) was run for all the categories of semen samples for their seminal plasma and the fertility associated protein was identified. A significant difference was found in the concentration of proteins in all subfractions when compared between control and infertile categories. Almost 86% of the protein was recovered from soluble fraction. In case of azoospermia, the protein content was very low when compared with fertile group. Seminal plasma proteins were visualized by silver staining. The molecular weight of the protein bands were ranging from 6.5 to 205 kDa. The band with molecular weight around 55 kDa was found to be missing in case of oligoasthenospermia. This particular protein is said to be fertility associated protein. The content of cholesterol for different subfraction of the human semen samples from infertile and fertile samples was compared. A wide range of cholesterol was recovered from prostasomes, that too purified. Conclusion: A thrive study have to be done in all the subfractions of the semen irrespective of the category of samples to know the exact function of the each subfractions in terms of protein and cholesterol distribution.