Aim: The main objective of this study is to compare and analyse human prostasomes amino acid content variation in normal men semen samples and infertile men semen samples for identification of clinical relevance. Materials and methods: Semen samples were collected from normal men (N=32) and from infertile men (N=32) and by following the standard world health organisation protocol semen analysis was done. Amino acid quantification was done by using amino acid analyzer. Prostasomes were separated from spermatozoa and seminal plasma by using centrifugation technique at 95000 RPM for 90 mins. Results: Independent sample T-test was carried out and shows that proline and alanine amino acids concentration (p<0.01) statistically significant compared with fertile men and infertile men. High concentration of amino acids in prostasomes were found in fertile men samples (18.09 ± 0.20 μmoles/L) when compared with infertile men samples (15.12± 0.37 μmoles/L). Conclusion: Amino acid in prostasomes plays an important role in the fertilization; the change in the concentration of amino acid in prostasomes leads to infertility of men. Here we found that the concentration of amino acids is high in fertile men when compared to infertile men which could act as an innovative diagnosis method for infertility.