Amrutharistam is a vital Ayurvedic formulation used for all kind of fever and rheumatic fever, which is prepared by fermenting the decoction of Tinospora cordifolia containing berberine as one of the active ingredient. Preliminary analysis indicated the presence of berberine being the largest concentration. The HPLC carried out in Lichrospher 100, RP-8e (250*4.6*5) column, mobile phase consisting of phosphate buffer and acetonitrile (70: 30). The determining wavelength was confirmed as 343nm. Five marketed formulations of Amrutharistam A-I, A-II, A-III, A-IV and A-V showed berberine concentration to be 9.63µg/ml, 10.10µg/ml, 10.20µg/ml, 9.64µg/ml and 10µg/ml respectively. The result showed the peak area response is linear within the concentration range of 10-50µg/ml with a correlation coefficient of 0.9998. Recovery studies 50%, 100% and 150% were conducted by standard addition method and found to be 99.804%. The developed and validated method can be effectively applied to the quantitative determination of berberine in Amrutharistam.