High-performance thin-layer chromatography (HPTLC) plays a crucial role in establishing the chemical fingerprint of natural products (NPs) during analysis. This technique involves standardized procedures for each step, ensuring the reproducibility of results. In this context, we present a comprehensive standard operating procedure (SOP) encompassing both manual instruments and suitable devices. The methods detailed herein adopt a non-targeted screening approach applicable to all unrelated samples, complemented by a two-step derivatization process capable of detecting a wide range of phytochemical classes. By following these procedures, researchers can obtain reliable and consistent results that could be used to build a database for NPs using HPTLC.