A simple, accurate, rapid and precise isocratic High performance liquid chromatographic (HPLC) method was developed and validated for the quantification of betaine in Achyranthes aspera. The method employs Reversed Phase Phenomenex (US) C18 Column 250 x 4.6mm I.D, 5µ Particle Size system consisted of prominence LC-20AT gradient pumps, Prominence SIL- 20A Auto sampler and a Prominence SPD-M20A Diode Array Detector (SHIMADZU), Japan and flow rate of 1.0 ml/min with a load of 20μl. Water and acetonitrile was used as mobile phase in the composition of 10:90. The detection was carried out at 242 nm. Linearity range was 0.32-0.48 mg/ml. Retention time of betaine was found to 8.42 min. The recovery studies 98-103%. This method was validated for accuracy, precision, linearity and Robustness as per ICH guidelines. The method was found to be specific, selective, and robust.