<jats:title>Abstract</jats:title><jats:p>A sensitive, selective, and robust high‐performance TLC (HPTLC) method using chiral TLC plates for qualitative and quantitative analysis of phyllanthin (<jats:bold>A</jats:bold>), hypophyllanthin (<jats:bold>B</jats:bold>), niranthin (<jats:bold>C</jats:bold>), and nirtetralin (<jats:bold>D</jats:bold>), the active lignans of<jats:italic> Phyllanthus </jats:italic>species, was developed and validated. The effectiveness and role of various stationary phases<jats:italic> viz</jats:italic> TLC silica gel 60F<jats:sub>254</jats:sub>, HPTLC silica gel 60F<jats:sub>254</jats:sub>, and chiral TLC plates in the quantitation were evaluated. A precoated chiral TLC plate was found suitable for the simultaneous analysis of four pharmacologically active lignans. For achieving good separation, the optimized mobile phase of <jats:italic>n</jats:italic>‐hexane/acetone/1,4‐dioxane (9:1:0.5 by volume) was used (<jats:italic>R</jats:italic><jats:sub>f</jats:sub> = 0.30, 0.36, 0.41, and 0.48 for compounds<jats:bold> A</jats:bold>,<jats:bold> B</jats:bold>, <jats:bold>C</jats:bold>, and <jats:bold>D</jats:bold>, respectively). A densitometric determination of the above compounds was carried out in reflection/absorption mode at 620 nm. Optimized chromatographic conditions provide well‐separated compact bands for the tested lignans. The calibration curves were found linear in the concentration range of 100–500 ng/band. Recoveries of<jats:bold> A</jats:bold>–<jats:bold>D </jats:bold>were 99.98, 100.51, 99.22, and 98.74%, respectively. The method was validated according to ICH guidelines. The method reported here is reproducible and applied for the quantitative analysis of the above lignans in the leaves of four <jats:italic>Phyllanthus</jats:italic> species,<jats:italic> i. e</jats:italic>., <jats:italic>P. amarus</jats:italic>, <jats:italic>P. maderaspatensis</jats:italic>,<jats:italic> P. urinaria</jats:italic>, and<jats:italic> P. virgatus</jats:italic>.