Xanthium spinosum is an annual plant commonly found in uncultivated and vacant areas. It is considered a quarantine weed because it reduces the value of crops and pastures. For therapeutic purposes, the aerial part of the plant is harvested be-tween June and August. Due to its phytochemical components, the plant exhibits numerous pharmacological activities, fo-cusing on antibacterial, anti-tumor, antitussive, antifungal, anti-inflammatory, antinociceptive, hypoglycemic, antimitotic, antioxidant, and diuretic effects, and it may be an effective treatment for contact dermatitis. The aim of this study was to determine the total and individual polyphenols in the alcoholic extract of Xanthium spinosum and to evaluate its biological and antiproliferative properties in vitro using tumor cell line models. Total polyphenols were determined by UV-VIS spec-trometry, while individual polyphenols were analyzed using LC-MS. The Caco-2 cell line was used for in vitro evaluation of cellular effects and therapeutic analysis of the Xanthium spinosum extract. The study results indicate that the 1% alcoholic extract of Xanthium spinosum has a cytotoxic effect on the Caco-2 cell line (colorectal adenocarcinoma) at concentrations of 500, 250, and 125 μg/ml, at both 48-hour and 72-hour exposure times, with a significantly lower proliferation rate observed at the 72-hour exposure.