We developed first HPTLC and HPTLC-MS/MS methods which enable characterization of structurally similar and complex biologically active compounds - physalins - from crude extracts of Chinese lantern (Physalis alkekengi L.). Separation on HPTLC silica gel plates developed with ethyl acetate-toluene-formic acid (7:3:0.2, v/v) enabled densitometric screening of physalins in absorption and, after post-chromatographic derivatization with sulfuric acid reagent, also in fluorescence mode. Compared to existing (U)HPLC methods, in this case TLC provides an alternative selectivity, better sensitivity and higher resolution, which was exemplified by the separation of physalin L standard and its impurity, identified as 2,3,25,27-tetrahydrophysalin A. Strong ion suppression caused by the developing solvent additive - formic acid - was efficiently solved by two successive plate pre-developments with methanol-formic acid (9:1, v/v) and methanol. This significantly improved the sensitivity of HPTLC-MS/MS method, but also required a slightly modified developing solvent ethyl acetate-toluene-formic acid (6:4:0.2, v/v). Simultaneous hyphenation of HPTLC with a triple quadrupole and an ion trap mass analyzer enabled a reliable and straightforward non-targeted characterization of physalins from the same chromatographic zone (band) and determination of physalin types. The performance of developed HPTLC-densitometric and HPTLC-MS/MS methods was demonstrated by the analysis of physalins from the aqueous extracts, prepared by an optimized fast and simple extraction method under reflux. Variations in physalin profiles and abundances in different parts of P. alkekengi L. harvested at different stages of maturity were observed. This indicates that not all parts of the plant, or plant as a whole, are appropriate for specific medicinal applications. Husks are proposed as the most suitable plant part for P. alkekengi L. quality control, because they exhibited the most obvious MS2 fingerprints of physalins with minimal interferences.