Citizens in developing countries rely on indigenous knowledge and practices and use locally available medicinal plants for different treatments. Due to limited instrumentation for chemical and biological characterization, most studies investigating the bioactive properties of Sri Lankan medicinal plants rarely progress to the molecular level. While for some plants, the whole plant extracts have been tested, most of the individual active compounds and their effect mechanisms have still not been identified. The separation of bioactive compounds from natural sources is a challenging task. Multi‐imaging high‐performance thin‐layer chromatography (HPTLC‐UV/Vis/FLD) combined with biological/biochemical assays (effect‐ directed analysis, EDA) and high‐resolution mass spectrometry (HRMS) provide the straightforward identification of natural products without prior tedious fractionation and compound isolation. Separating complex plant extracts into individual compounds, and still on the same adsorbent surface, studying their effects highlighted their potential. These findings can be used to substantiate current traditional medicinal knowledge and to evaluate their benefits, risks, and limitations. The developed hyphenated HPTLC‐UV/Vis/FLD‐EDA‐HESI‐ HRMS methods for identification of single compound effects in Sri Lankan Abelmoschus moschatus and Cinnamomum zeylanicum included (1) non‐target screening, (2) assignment of prominent individual bioactive compounds, and (3) comparison of product profiles to check the quality of commercial products. Both studies revealed not only the phytochemical profiles but also prioritized bioactive constituents. Diverse antimicrobials, antioxidants, and inhibitors of glucosidase, tyrosinase, and cholinesterase were detected. Running reference standards in parallel to identified compounds, confirmed the assignments and bioactivities. The sustainable and environmentally friendly technique can be used in developing countries for profiling and valorization of plant‐based preparations.