A new validated high-performance thin-layer chromatographic (HPTLC) method has been developed for the simultaneous determination of anti-inflammatory compounds betulinic acid (BA, 1), 24β-ethylcholesta-5,22E,25-triene-3β-ol (ECTO, 2), and lupeol (LU, 3) in the roots of Clerodendrum phlomidis. Extraction efficiency of marker compounds was studied using normal (cold and hot), ultrasonic, as well as microwave-assisted extraction techniques with various solvents. Well-resolved separation of marker compounds was achieved on silica gel 60F254 plates using the mobile phase consisting of chloroform-methanol (98:2, ½/½). Marker compounds were scanned using the densitometric reflection-absorption mode after post-chromatographic derivatization with vanillin-sulfuric acid reagent. Validation of method was performed as per the International Conference on Harmonization (ICH) guidelines. Report on the occurrence of betulinic acid for the first time in C. phlomidis is of chemotaxonomic importance. In addition, anti-inflammatory potential of the rare sterol ECTO (2) on lipopolysaccharide (LPS)-stimulated production of pro-inflammatory cytokines (tumor necrosis factor-α [TNF-α] and interleukin-6 [IL-6]) was also evaluated as it was not reported earlier.