Pepper is one of the most consumed spices in the world. Its main compound is capsaicin, a widely studied biomarker that has pharmacological activities due to its anti-inflammatory and analgesic capacity. Topical formulations such as patches based on capsaicin have been developed as an option in relieving pain and reducing swelling. In addition, capsaicin is used as an active ingredient in non-lethal weapon formulations such as pepper spray through the QuEChERS concept (Quick, Easy, Cheap, Effective, Robust, Safe) technique. Used for food analysis, it allows the direct analysis of the biomarker by TLC-ESI-MS, which are previously separated by HPTLC (High Performance Thin Layer Chromatography) using an internal standard for determination of Rf and confirmation of capsaicin in different matrices.