Over the past two decades, concerns have arisen about the efficacy and safety of medicinal plants, highlighting good agricultural practices and quality control. This study aimed to validate a multi-residue analysis method for detecting 268 pesticides in Mikania glomerata tincture, using direct injection and UPLC-MS/MS, per SANTE guidelines. The validation of the method involved evaluating the linearity of analytical curves in terms of the determination coefficient r2 and residuals (%), as well as the limits of quantification (LOQ), matrix effects, precision (expressed as the coefficient of variation, CV), and accuracy (determined as the recovery percentage). The parameters and acceptance criteria were assessed based on SANTE guidelines. The method was then applied to analyse commercial samples of M. glomerata tincture, and traces of carbendazim and dimethomorph were detected. This study underscores the need for regulatory measures to enhance agricultural practices, thereby ensuring the safety and efficacy of medicinal plants.