Particularly in the last decades, antioxidants have received a lot of attention in science and the general public regarding their roles as nutrients with potential health benefits and as important biomolecules. Apart from universal analytical methods, several different assays and countless modifications of these assays exist to analyse total antioxidant capacity (TAC) in vitro. One of the most popular is the DPPH assay, which is based on the stable radical 2,2-diphenyl-1-picrylhydrazyl and its reaction with radical scavengers. Normally, this reaction is observed in the spectrophotometer at a wavelength of 517 nm, but the method has been successfully combined with HPLC and thin layer chromatography in the past. A substantial body of work exits on the mechanisms, interactions and possible influences in the traditional photometric assay. However, unique effects of the stationary phase on the radical reaction have been reported. The aims of this study are to evaluate the existing information on the HPTLC-DPPH method, to determine the effects of various parameters of the method and optimize them to develop a reproducible approach for antioxidant analyses. Kinetics, precision, and calibration of the method as well as possible sources of error were also examined to certain extend.Particular focus was put on the analysis of different herbs and spices as well as on green tea catechins.