The objective of present study is to develop and validate a simple, precise, rapid, specific and accurate reverse phase HPLC method for the simultaneous determination of daidzein, genistein, formononetin and biochanin A obtained from in vitro cultured cells of Trifolium pratense L. Chromatographic separation was achieved on reverse phase octadecylsilane C18 column with isocratic elution. The mobile phase consisted of a mixture of ammonium acetate and methanol (40:60) pumped at a flow rate of 1.0 ml. min-1. The effluents were monitored at 254 nm with a UV detector at ambient temperature. Validation was carried out according to International Conference on Harmonisation (ICH) guidelines with respect to linearity, sensitivity, accuracy, precision, robustness and ruggedness. Good linearity was observed (r2 > 0.998) over the test concentration range of the four isoflavones. Precision, accuracy, limit of detection and limit of quantification obtained were within acceptable range in each case. The method was robust and specific for the four isoflavones. The results indicate that the developed method is simple, precise, rapid, specific and accurate, and would be suitable for the quantification of daidzein, genistein, formononetin and biochanin A obtained from in vitro cultured cells of Trifolium pratense L.