A selective, specific, precise, linear, accurate and robust analytical method was developed and validated for the assay of vancomycin HCl in vancomycin hydrochloride injection. Comparative UV spectrophotometric and reverse‐phase HPLC were used to develop the quantitative determination. Acetonitrile and pH 2.2 phosphate buffer in the ratio 20:80 v/v were used as the mobile phase, and a flow rate of 1.0 ml/min with a 20 min run time. The detection was carried out at 235 nm with a Nucleosil C18 (250 × 4.6 mm) 10 μm column, and the ambient column temperature was maintained. The method uses a 20 μl injection volume and diluent as a blank solution in this connection. The method was validated as per the current regulatory guidelines. The linearity of this method was found to be linear in the range of 50–150% of the working concentration, and the correlation coefficient was >0.999. The method’s accuracy was within the acceptable range, which was 98.1–101.5%. The method’s precision was within an acceptable range of about 0.32% RSD. The analytical solution was stable for up to 48 h at room temperature. The method’s robustness was proved by utilizing quality design tools. Stress studies demonstrated the method’s stability‐indicating nature.