Pesticides are major contaminating chemicals in agricultural environment and hazard to exposed population. The pesticides form a strong class of environment pollutants, as they are sometimes nonbiodegradable, damaged not only the environment and agriculture but also they have entered into the food chain thereby affecting health and development. Malathion is a broad spectrum organophosphorous insecticide generally used to control a variety of insects. The present study was planned to develop a new method for analysis of malathion in human blood samples using thin layer chromatography technique, which is simple and quick. Malathion was extracted fromblood using solvent extraction methods and then identified on TLC plates. For chromatographic separation various binary and tertiary solvent systems were used and for detection, on developed plates, palladium chlorides reagent was used which successfully increased the sensitivity without dispensingwith simplicity of themethod. Statistical analysis was performed on four solvent system namely benzene: methylen dichloride [80:20], hexane: ethyl acetate [50:50], hexane :ethyl acetate [20:80], and hexane: propanol [20:80] which include the calculation of mean Rfvalue, value of standard deviation and co-efficient of variance. It is evident from the statistical data that hexane: ethyl acetate[50:50] and hexane :ethyl acetate [20:80] are preferably good solvent systems formalathoin identification by thin layer chromatography.