<p><i>Solanum nigrum</i> Linn., a species belonging to the Solanaceae family, possesses a documented history of ethnobotanical application, with its berries utilized as both a nutritional resource and a therapeutic remedy. The berries are rich in a diverse array of phytoconstituents, notably steroidal glycoalkaloids, polyphenols such as flavonoids and tannins, coumarins, saponins, proteins, carbohydrates, glycosides, and phytosterols. The current research focuses on the development of a robust and precise high-performance thin-layer chromatography (HPTLC) method for the simultaneous quantitative determination of quercetin and solasodine in <i>S. nigrum</i> Linn. berries. The method was validated as per the International Council for Harmonisation (ICH) Q2(R2) guidelines. The separation was achieved using pre-coated aluminum silica gel 60 F₂₅₄ plates as the stationary phase and toluene‒ethyl acetate‒formic acid (5:4:1, <i>V/V</i>) as the mobile phase. The <i>R</i>_F value of quercetin and solasodine were found to be 0.63 and 0.23, respectively. The densitometric scanning was performed at 382&#xa0;nm for quercetin and at 596&#xa0;nm for solasodine after derivatization with anisaldehyde‒sulfuric acid reagent. Linearity was demonstrated for quercetin and solasodine across a concentration range of 200–1200&#xa0;ng/band with correlation coefficients (<i>r</i>²) 0.9967 for quercetin and 0.9978 for solasodine. The limits of quantification for quercetin and solasodine were found to be 163&#xa0;ng/band and 140&#xa0;ng/band, respectively, whereas the limits of detection for quercetin and solasodine were 54&#xa0;ng/band and 47&#xa0;ng/band, respectively. The developed method demonstrated robustness, precision, accuracy, and specificity for the quantification of quercetin and solasodine, both in bulk form and within the hydroalcoholic extract of <i>S. nigrum</i> Linn. berries.</p>