The present study Estimation of drugs in biological media is increasingly important nowadays, which reveals information like bioavailability, bioequivalence, drug abuse, and pharmacokinetics and drug research. HPLC is the most suitable technique for the analysis of biological fluids owing to its well-developed characteristics and ruggedness. It is an extremely sensitive, precise, accurate, and rapid, separation technique. Clotrimazole is a broad spectrum antifungal agent that is used for the treatment of infections caused by various species of pathogenic dermatophytes, yeasts, and Malassezia furfur. The primary action of Clotrimazole is against dividing and growing organisms. Clotrimazole interacts with yeast 14-α demethylase, a cytochrome P-450 enzyme that converts lanosterol to ergosterol, an essential component of the membrane. In this way, Clotrimazole inhibits ergosterol synthesis, resulting in increased cellular permeability. Few analytical methods were reported for the quantitative determination of Clotrimazole and its combination with other drugs by HPLC. Only one method was reported for Clotrimazole in mice plasma by capillary electrophoresis. But there is no RP-HPLC method reported for the bio-analytical method development in human plasma using protein precipitation method under different chromatographic conditions. The present study is planned to develop newer analytical method for the determination of Clotrimazole by bio-analytical method development in human plasma using protein precipitation by RP-HPLC under different chromatographic conditions. CONCLUSION: A bioanalytical method was developed for the estimation of Clotrimazole by HPLC method. The method was validated for its transferability to other user or other laboratory. The HPLC method was developed by using 0.5%TEA in water (pH 3 adjusted with Orthophosphoric acid) and Acetonitrile in meticulous ratio. The peaks obtained for the drugs of interest by the present method are well resolved from each other without any interference and from the plasma endogenous proteins. The peaks are symmetrical with acceptable tailing factor. The retention time of Clotrimazole was within the limit. The results of linearity, intraday and interday precision study and capability of the extraction method were within the limits of bioanalytical method development. The method was linear with a correlation coefficient of acceptable agreement, which is suitable for the estimation of Clotrimazole in human plasma and other biological fluids. The method demonstrated relative recoveries with acceptable relative standard deviation. The limit of quantification (LOQ) and limit of detection (LOD) for Clotrimazole was found to be nanograms lesser than unity. Hence the developed method is sensitive for the estimation of Clotrimazole in trace amounts. Peak purity studies, with peak purity index values closer to unity reveals that the method developed was specific for the estimation of Clotrimazole in blood and other biological fluids. It can be concluded that the developed RP-HPLC method in human plasma was found to be very simple, reliable and selective for providing satisfactory accuracy and precision. The methods are suitable for routine quantitative analysis in pharmaceutical dosage forms. Hence this developed method can be used further in Bioequivalence and bioavailability studies of clotrimazole. Pharmacokinetic and bio-equivalence study centers