<jats:title>Abstract</jats:title><jats:p>A sensitive, selective, precise, and robust high‐performance thin‐layer chromatography method was developed and validated for analysis of two new recently isolated sterols, 4α‐methyl‐24β‐ethyl‐5α‐cholesta‐14,25‐dien‐3β‐ol (<jats:bold>1</jats:bold>) and 24β‐ethylcholesta‐5,9(11),22<jats:italic>E</jats:italic>‐trien‐3β‐ol (<jats:bold>2</jats:bold>), and a triterpene, betulinic acid (<jats:bold>3</jats:bold>), in<jats:italic> Clerodendrum inerme</jats:italic> extract. The method employed HPTLC plates precoated with silica gel 60F<jats:sub>254 </jats:sub>as the stationary phase. To achieve good separation, an optimised mobile phase consisting of toluene‐acetone (94:06, v/v) was used (<jats:italic>R</jats:italic><jats:sub>f </jats:sub>0.48, 0.34, and 0.22 for compounds<jats:bold> 1</jats:bold>, <jats:bold>2</jats:bold>, and<jats:bold> 3</jats:bold>, respectively). Densitometric determination of the above compounds was carried out in reflection/absorption mode at 620 nm. Optimised chromatographic conditions provide well separated compact spots for the compounds<jats:bold> 1</jats:bold>,<jats:bold> 2</jats:bold>, and<jats:bold> 3</jats:bold>. The calibration curves were linear in the concentration range of 100–2500 ng/spot. The method was validated for precision, robustness, and recovery. The limits of detection and quantitation were 5, 6, and 10 μg/mL and 14, 18, and 29 μg/mL, respectively, for <jats:bold>1</jats:bold>, <jats:bold>2</jats:bold>, and <jats:bold>3</jats:bold>. The method reported here is reproducible and convenient for quantitative analysis of these compounds in the aerial parts of<jats:italic> C. inerme</jats:italic>.