Croton antisyphiliticus Mart. is a plant popularly used in folk medicine by traditional communities from Brazilian Cerrado savanna to treat general inflammation. According to ethnopharmacological employment, this specie can be considered a source of biologically active molecules for the development of antinociceptive and anti-inflammatory drugs. Thereby, this study reports the dereplication approach of C. antisyphiliticus roots and the in vivo evaluation of the antinociceptive and anti-inflammatory potential with albino Swiss mice. Based on HPLC coupled to Q-Exactive Orbitrap Mass Spectrometer and using GNPS, it was annotated thirteen polyphenolics compounds, being four reported for the first time in the Croton genus. Treatment in vivo with ethanolic and aqueous roots extracts inhibited the number of writhes in a dosedependent manner, reduced the pain induced by formalin and hyperalgesia induced by carrageenan. These extracts also reduce paw oedema, cell migration and the myeloperoxidase activity, with effects similar to the indomethacin and dexamethasone drugs.