The study aims to analyze the fragmentation patterns of cannabidiol (CBD) bio-derivatives using High-Performance Liquid Chromatography coupled with Tandem Mass Spectrometry (HPLC-MS/MS). By employing soft ionization techniques (ESI and APCI), the research focuses on identifying structural integrity and cleavage pathways of three specific CBD derivatives. This approach enhances understanding of their breakdown mechanisms, which is crucial for optimizing their pharmacokinetic properties and therapeutic potential. The study underscores the role of structural modifications, like Mannich-type reactions, in improving CBD’s solubility, stability, and efficacy for medical applications.