A sensitive, selective, and robust high-performance TLC (HPTLC) method using chiral TLC plates for qualitative and quantitative analysis of phyllanthin (A), hypophyllanthin (B), niranthin (C), and nirtetralin (D), the active lignans of Phyllanthus species, was developed and validated. The effectiveness and role of various stationary phases viz TLC silica gel 60F(254), HPTLC silica gel 60F(254), 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 n-hexane/acetone/1,4-dioxane (9:1:0.5 by volume) was used (R(f) = 0.30, 0.36, 0.41, and 0.48 for compounds A, B, C, and D, 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 A-D 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 Phyllanthus species, i. e., P. amarus, P. maderaspatensis, P. urinaria, and P. virgatus.