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A new method was established for simultaneous estimation of Kanamycin and Cephalexin by RP-HPLC method. The chromatographic conditions were successfully developed for the separation of Kanamycin and Cephalexin by using symmetry C18 column (4.6×150mm) 5µ, flow rate was 1ml/min, mobile phase ratio was (70:30 v/v) methanol: phosphate buffer (KH2PO4 and K2HPO4) pH 3 (pH was adjusted with orthophosphoric acid), detection wave length was 258nm. The instrument used was WATERS HPLC Auto Sampler, Separation module 2695, photo diode array detector 996, Empower-software version-2. The retention times were found to be 2.403 mins and 3.907 mins. The % purity of Kanamycin and Cephalexin was found to be 100.27% and 99.87% respectively. The system suitability parameters for Kanamycin and Cephalexin such as theoretical plates and tailing factor were found to be 2294, 1.27 and 4891 and 1.03, the resolution was found to be 8.67. The analytical method was validated according to ICH guidelines (ICH, Q2 (R1)). The linearity study n Kanamycin and Cephalexin was found in concentration range of 50µg-250µg and 5µg-50µg and correlation coefficient (r2) was found to be 0.999 and 0.999, % recovery was found to be 99.56% and 99.48%, %RSD for repeatability was 0.27 and 0.40, % RSD for intermediate precision was 0.27 and 0.94 respectively. The precision study was precise, robust, and repeatable.LOD value was 2.17 and 6.60, and LOQ value was 0.032 and 0.1125 respectively.