<p>A high-performance thin-layer chromatography (HPTLC) method for fingerprint profiling and quantitative estimation of stigmasterol was developed and validated in <i>Syngonium wendlandii</i> Schott leaf extracts. Ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit gene (rbcL) DNA barcoding supported plant identification. Optimized mobile phases were used for chromatographic separation on silica gel 60 F₂₅₄ plates of selected phytochemical standards. Stigmasterol was quantified densitometrically at 416&#xa0;nm. The technique was validated in accordance with the International Conference of Harmonization’s (ICH) guidelines and demonstrated linearity over the range 10–30&#xa0;µg/spot (<i>r</i> = 0.9978). Limit of detection and limit of quantification were 1.89 and 5.74&#xa0;µg/spot, respectively. Precision studies showed percent relative standard deviation (%RSD) values below 2%, with recovery rates ranging from 99.3% to 103.5%. The extracts of petroleum ether (19.0 ± 1.6&#xa0;µg/g) and chloroform (16.6 ± 1.3&#xa0;µg/g) showed higher stigmasterol levels, as determined by quantitative analysis. HPTLC fingerprinting revealed solvent-dependent differences in sterol, flavonoid, and alkaloid profiles. Gas chromatography–mass spectrometry (GC–MS) examination of the hydroalcoholic extract revealed the presence of phenolic derivatives, fatty acid esters, terpenoid-related compounds, and phytosterols, while Fourier transform infrared spectroscopy (FTIR) spectra showed hydroxyl, aliphatic, and carbonyl functionalities. The methanolic and hydroalcoholic extracts had higher total phenolic content and stronger in vitro antioxidant activity, with 1,1-diphenyl-2-picryl-hydrazil (DPPH) IC₅₀ (half maximal inhibitory concentration) values of 72.4 ± 0.04 and 49.1 ± 0.16&#xa0;µg/mL, respectively. The study presents preliminary chromatographic and phytochemical data that could be beneficial for future standardization and research into <i>S. wendlandii</i> leaf extracts.</p>