Despite the fact that Thymus serpyllum is well-known medicinal plant and its chemical profile and biological activity have been investigated, there is no detailed study regarding the influence of different techniques and conditions on the extraction of polyphenolic compounds from Serpylli herba. The aim of this study was optimization of the extraction parameters that improves the efficiency of polyphenols extraction from T. serpyllum: particle size, solid-to-solvent ratio, solvent type and extraction time, by using maceration, heat- and ultrasound-assisted extraction (HAE and UAE). The extraction efficiency was expressed via total polyphenol content (TPC) and total flavonoid content (TFC). The statistical analysis (one-way ANOVA and full factorial design) has revealed that the optimal conditions for achieving the best polyphenols yield were particle size of 0.3 mm, 1:30 solid-to-solvent ratio and 50% ethanol, as environmentally friendly extraction medium, while extraction time has not shown statistically significant influence on polyphenols concentration, in all procedures. Under these conditions, the measured TPC was 26.6 mg GAE/L in maceration, 29.8 mg GAE/L in HAE and 32.7 mg GAE/L in UAE, which was in agreement with the predicted values, while TFC was 143 mg CE/L, 12.4 mg CE/L and 16.7 mg CE/L for maceration, HAE and UAE, respectively. According to total polyphenols yield, the efficiency of the extraction methods for all variables was ranked by significance in the following order: UAE > HAE > maceration, whereas total flavonoids yield was the highest in UAE, although there was no statistically significant difference between maceration and HAE. According to our results, UAE could be selected as the most successful and suitable technique for extraction of bioactive polyphenolic compounds from Serpylli herba. Using LC/MS and HPLC analysis, 9 polyphenolic compounds were identified and quantified: 6,8-Di-C-glucosylapigenin, chlorogenic acid, 6-hydroxyluteolin 7-O-glucoside, caffeic acid, luteolin 7-O-glucuronide, apigenin glucuronide, salvianolic acid g isomer, rosmarinic acid and salvianolic acid I. This study was an initial step in production of polyphenols-rich wild thyme extracts aimed to be used for formulation of foodstuffs and medicines. (C) 2017 Elsevier B.V. All rights reserved.