An efficient micropropagation protocol was developed for the endangered medicinal plant species, Exacum bicolor Roxb. (Gentianaceae) using nodal segments by producing axillary shoots. Quantitative estimation of active compounds of medicinal properties such as luteolin and chlorogenic acid (flavonoids) was made in in vitro regenerated plantlets and compared to that of the wild intact plant by HPLC analysis. The results of the study revealed that the luteolin content was determined to be significantly higher in the wild plant tissues than the plantlets regenerated. However, this trend was reverse for the content of chlorogenic acid.