High-performance thin-layer chromatography (HPTLC) is a powerful separation technique which is often overlooked. In this study, we comprehensively assessed the applicability, ease, and performance of HPTLC in combination with densitometry and mass spectrometry (MS) to characterize physalins — relatively polar secondary metabolites from Physalis alkekengi L. HPTLC silica gel plates were evaluated in combination with 14 developing solvents (13 published in the literature). Bonded stationary phases (HPTLC RP-18, RP-18 W, CN F254S) were also tested. Four detection reagents (sulfuric acid, anisaldehyde, 4-dimethylaminocinnamaldehyde (DMACA), and molybdatophosphoric acid) were compared to ascertain which one is the most suitable. For all chromatographic analyses, a commercial standard physalin L and a P. alkekengi L. crude extract were used. In some cases, physalin L standard appeared as two clearly resolved bands on silica plates, but only after derivatization, where sulfuric acid reagent provided the best sel...