During the present study an analytical method based on reverse-phase high performance liquid chromatography photodiode array detection method was developed for simultaneous determination of withaferin-A and withanolide-A in plant parts of two cytotypes (diploid n=12 & tetraploid n=24) of Physalis angulata. All the plant parts were extracted in different solvent solutions i.e., acidic [HCl] methanol, methanol, n-hexane, chloroform. Both the compounds were comparatively analyzed. The results revealed that tetraploid cytotype (n=24) showed the higher composition of both the reference compounds. The HPLC separation process was performed on Attantis T3 Column (5µm pore size, 4.6mm×250mm) with diode-array detection held at 25°C utilizing, acetonitrile and water as mobile phase at flow rate of 0.8mL/min in isocratic elution. Calibration curves produced satisfactory linear regression (r 2 >0.999) for each examined sample. The method is simple, rapid and provides better resolution can be easily applied to the quantitative analyses of withanolides in plant matrices.