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 (<2) indicates that the method is precise and accurate. The robustness and ruggedness of the proposed methods were studied on two different wavelengths with the help of analysts. The UV-spectrophotometric method was found to be secure and useful for the calculation of microsphere-loaded Cyclophosphamide.The novelty of this research is based on a safe, simple, and effective method thatis cost-effective and time-saving for the validation of cyclophosphamide.