Cardiac glycosides are the secondary metabolites that are used in atrial fibrillation and atrial flutter as it binds with the sodium-potassium ATPase pump and increases the force of contraction. There are two types of cardiac glycosides i.e. cardenolides and bufadienolides. Digoxin is a cardenolide type of cardiac glycoside. Digoxin is found in chloroform extract of Moringa concanensis leaves. In the present study, the reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated to determine the content of digoxin in chloroform extract of M. concanensis leaves from three different geographical sources. The chromatographic method was developed at 220 nm wavelength with an Acetonitrile: Water: 1% Orthophosphoric acid (50:50:0.1 %V/V/V) mobile phase. In this method, the digoxin shows linearity in the range of 10 – 50 μg/ml with a regression coefficient of 0.995. The Limit of detection (LOD) and Limit of quantitation (LOQ) was found to be 0.232 μg/ml and 0.703 μg/ml respectively.