The present study was aimed to identification, isolation, and quantification of marker in R. tuberosa (Acanthaceae). HPTLC fingerprinting was carried out for various extract of root, stem, and leaf of R. tuberosa. From the HPTLC fingerprint the florescent band (under 366 nm) at : 0.56 (mobile phase chloroform : toluene : ethyl acetate (6 : 3 : 1, v/v)) was found in leaf, root, and stem of R. tuberosa. So, the florescent band (under 366 nm) at : 0.56 was isolated as marker compound RT-F2 from root of R. tuberosa. The marker compound RT-F2 was quantified by using HPTLC technique. The percentage (W/W) amount of RT-F2 was found to 40.0% and 44.6% in petroleum ether and ethyl acetate extract of R. tuberosa roots, respectively. Further study is suggested to characterization and biological nature of marker compound.