A novel, accurate, rapid and cost effective quantification method was developed for Withaferin A in herbal combined dosage forms using a destructive reagent. The separation was carried out using mobile phase of toluene : ethyl acetate : formic acid (5 : 5 : 3 v/v/v) at a detection wavelength of 440 nm. The method was validated as per the ICH guideline. The calibration range was found to be 90–630 ng per band. The method was found to be accurate in triplicate results at different standard addition levels. The LOD and LOQ were 2.59 and 7.87 ng respectively. The percentage relative standard deviation was less than 2 for the system suitability testing. The efficiency of the method for separation and quantification was confirmed by applying the same method, without modification, to 11 formulations.