A high-performance thin-layer chromatographic (HPTLC) method for the determination of the two main potato glycoalkaloids, alpha-chaconine and alpha-solanine, in different parts of the potato plant, is described. Samples were extracted with diluted aqueous acetic acid containing the ion-pairing reagent 1-pentanesulfonic acid, sodium salt. Extracts were purified and analytes fivefold concentrated by a solid-phase extraction (SPE) procedure using a Sep-Pak C18 cartridge. The methanol SPE eluate was applied to the HPTLC silica gel 60 F254 plates. The separation was performed in chloroform-methanol-2% aqueous NH4OH (70:30:5) mixture as the mobile phase. Six different detection reagents: two modifications of the Dragendorff's reagent, phosphomolybdic acid, paraformaldehyde-phosphoric acid, Ce(IV)sulfate-sulfuric acid and sulfuric acid-ethanol reagents were applied for quantification by densitometry. Fluorescence enabled detection of 10 ng of each alkaloid.