The leaves, root and bark of the aromatic African indigenous plant, Croton gratissimus Burch. (Euphorbiaceae), are widely used in traditional medicine to treat coughs, chest complaints, rheumatism, abdominal disorders and fever among others. In Afrikaans it is referred to as "Koorsbessie" which alludes to its traditional use as a pyrogenic. The chemical composition of plants is very complex and analysis and quality control can be very challenging due to natural variability. In addition, very few reference standards from plants, especially from Africa, are commercially available. Due to its visual nature and the holistic fingerprint produced, high performance thin layer chromatography (HPTLC) is often recommended for the quality control of plant material. The aim of this study was to develop an HPTLC fingerprint and isolate marker compounds for inclusion on HPTLC plates to enable quality control. Croton gratissimus leaf samples were collected from various parts of South Africa and extraction was optimised. HPTLC fingerprints were developed and optimised and images were captured before and after derivatisation under UV (254 nm, and 366 nm) and white light. Preparative high performance liquid chromatography hyphenated to mass spectrometry (HPLC-MS) was used to isolate marker compounds. Method development and optimisation determined the following: most efficient extraction solvent = methanol:water (8:2 v/v); mobile phase = ethyl acetate:acetic acid:formic acid:water (100:11:11:27 v/v/v/v); and derivatising agent = natural product reagent. UPLC-MS analysis and 1D NMR spectroscopy were used to characterise and identify compound 1 as isoorientin and compound 2 as kaempferol-3-β-D-(6″-O-trans-p-coumaroyl) glucopyranoside, which correlated well with published spectral data. The final HPTLC fingerprint with biomarkers included showed good separation for profiling purposes and well-defined bands. The biomarkers at retention factor (Rf) 0.30 and Rf 0.69 for isoorientin and kaempferol-3-β-D-(6″-O-trans-p-coumaroyl) glucopyranoside, respectively, were present in all samples but varied quantitatively. The HPTLC method developed provided a good fingerprint for species authentication. Preparative HPLC-MS played a major role in successfully isolating marker compounds to be used for the quality control of C. gratissimus.