The creatine/phosphocreatine system is essential for cellular phosphate coupled energy storage and production, particularly in tissues subject to high metabolic demands. Male factor infertility is a common condition with unknown etiology in most of the cases. Sperm abnormalities could possibly lead to infertility. As sperm motility depends on intact mitochondrial function and energy levels. Thus reduced intracellular creatine stores may contribute to decreased sperm motility leading to male infertility as creatine /phosphocreatine system plays major role in making and breaking of ATP, thus in energy kinetics. We developed and validated a denaturing high performance liquid chromatograph (DHPLC) method for the molecular analysis of SLC6A8 and GAMT genes involve in creatine biosynthesis and transport as a possible source of human male infertility by analyzing DNA from 64, clinically confirmed, infertile men. No mutation/polymorphism was detected in the exonic regions of both genes in all the patients and in fertile healthy controls indicating that SLC6A8 and GAMT genes may not be directly involved in human male infertility.