This paper presents a simple and rapid approach to the quantification of various glycosides using high-performance thin-layer chromatography (HPTLC). Different classes of glycosides, represented by genistin and ononin (both monosaccharidic O-glycosides), rutin (a disaccharidic O-glycoside) and luteolin-6-C-glucoside (a monosaccharidic C-glycoside), were successfully separated using a mixture of ethyl acetate‒methanol‒glacial acetic acid‒formic acid (11:1:1:1, V/V) as the mobile phase followed by derivatisation with natural product–polyethylene glycol (NP–PEG) reagent. The method was validated for the quantification of these glycosides in accordance with the guidelines of the International Council for Harmonisation. The general applicability of the validated approach is demonstrated with the analysis of a large number of glycosides including two glycosides (i.e. rutin, naringin) in commercial products.