Cyclophosphamideis a chemotherapy medication used to treat cancer. It is also used to suppress the immune system. The present investigation deals with the preparation of microspherescontaining cyclophosphamide. The method has been developed and validated for the estimation of cyclophosphamide using water as a diluent. The λ max of cyclophosphamide in water was found to be 263 nm. The parameters, range, linearity, accuracy, precision, LOD, LOQ, robustness, and ruggedness were determined. The drug cyclophosphamide showed linearity in the concentration range from 0.4-1.4μg/ml with a correlation coefficient value of 0.999. The proposed method was applied to pharmaceutical formulation and the percentage amount of drug estimated was found to be 99.3%. The accuracy of the method was checked by a percentage recovery experiment at three levels, i.e., 80%, 100%, and 120%. The percentage recovery was found to be in the range of 99±100%. The precision of the method was studied as day variation. The % RSD value within the official limit (