Normal-Phase High Performance Liquid Chromatography method was developed for determination of Rapamycin, Temsirolimus Regio isomer (monoester) and Temsirolimus diester in Temsirolimus drug and pharmaceutical formulations. The separation was accomplished on YMC Pack SIL (250 x 4.6 mm, particle size 5μm) column under isocratic mode. The column oven temperature was set at 35° C and Auto sampler temperature used at 5°C. The mobile phase is a mixer of n-hexane, ethanol and trifluoroacetic acid in the ratio of 90:10:0.01 and PDA detector set at 280 nm used for detection. The retention times of Rapamycin, Temsirolimus peak, Temsirolimus monoester and Temsirolimus diester peaks are 12.29, 21.24, 33.11 and 51.91 min. respectively. The method has been fully validated and is linear. Results of analysis are validated statistically and by recovery studies. During the process development of Temsirolimus, three process impurities (Rapamycin. Temsirolimus monoester and Temsirolimus diester) are detected by high performance liquid chromatography (HPLC). This method offers advantages over using photodiode-array UV detection (LC-PDA) for the determination of peak purity, namely components with similar UV spectra can be distinguished, the molecular mass of the impurity can be determined, and structural data can be obtained by using LC-MS. The result of studies shows that the proposed normal phase-HPLC method is found to be precise, linear, accurate, rugged, selective, specific, and robust. Hence this method may be used for the routine analysis in bulk drug and in its pharmaceutical dosage forms.