► Simple TLC-DPPH method was standardized. ► Influence of adsorbent type, time and solvent of derivatizing agent solution was examined. ► Antioxidant activity of selected natural polyphenolics was investigated. TLC-DPPH test belongs to a group of frequently performed assays aimed at detection of compounds with desired activity (effect directed analysis). Despite its popularity a standard procedure has not been elaborated so far causing difficulties in comparing results obtained in different laboratories. Thus the aim of the presented research was an approach to develop a standardized procedure for assessing free radical scavenging properties of plant polyphenols. It was observed that specifically positive adsorbent (silica gel) strengthened the observed result of radical-antioxidant reaction, while polar bonded stationary phase CN-silica weakened it. Based on the observed results it was concluded that the TLC-DPPH assay should be preferably performed on the surface of non-specific adsorbents (e.g.: RP-18) with the use of n-hexane for DPPH dissolution. It is also proposed to document the results every 5min after staining, as they change in time.