A Novel stability-indicating method was developed and validated by Reverse phase Hplc method   for the estimation of Thiotepa in Lyophilized form. The simple, Accurate Assay method was developed by PDA Detector at 215nm and column(YMC,150mmx4.6mm, 3.5 μm,12nm).The mobile phase A consisted of (3.5g of potassium Dihydrogen phosphate/1liter: 4.2g of Disodium hydrogen Phosphate/2liters)Buffer : Acetonitrile (85:15v/v) and The mobile phase B consisted of (3.5g of potassium Dihydrogen phosphate/1liter: 4.2g of Disodium hydrogen Phosphate/2liters)Buffer : Acetonitrile (50:50v/v) gradient flow rate at 1.0 mL/min for 30minutes. Samples was stored at 5°c temperature in sample cooler and Column oven temperature was maintained  in the method at 30°c  Respectively. The retention time of Thiotepa was 11.313 minutes. The proposed method was developed and validated with respect to specificity, accuracy, precision, linearity, Analyte Stability. Specificity of the Assay method shows no interference from Diluent and degrading products formed by alkaline, acidic, oxidative, Water Hydrolysis conditions. At Each level, %Recovery of Thiotepa in formulations was obtained to be in a range of 98-102%. The linearity of the assay was established in the range of 159.915-479.744µg/mL with correlation coefficient (R2) > 0.9999. This method was shows simple, precise, more accurate, stability indicating and reliable determination of Thiotepa for drug stability assay in pharmaceutical studies.