Diarrhoea is a leading killer of children, accounting for 9% of all deaths among children under age 5 worldwide and 3% in Malaysia in 2015. A large proportion of diarrhoea illnesses among children in developing countries are ascribed to an unknown etiology because microscopic examination was the only available technique which has low detection limits. The proposed study aimed to evaluate a new quadriplex PCR assay to detect parasitic pathogens namely E. histolytica, G. lamblia and C. parvum which considered responsible for the majority of human infections. Three set of specific primer pairs were designed for detection of parasitic pathogens. Quadriplex PCR assay was optimized and an internal amplification control was incorporated to check for PCR inhibitors in samples. The PCR assay was evaluated using spiked stool samples. Specific primer pairs were successfully designed and simultaneously amplified the targeted genes. The analytical sensitivity of the quadriplex PCR at the DNA level was found to be 50 ng DNA. The analytical specificity was evaluated with 11 reference protozoal and bacterial strains and was found to be 100%. We concluded that the developed quadriplex PCR assay was rapid and gave results within 5 hours which is essential for the identification of parasitic pathogen and might be useful as an additional diagnostic tool whenever time is important in the diagnosis of parasite that cause diarrhoea.