Avocatin B (AVO) is an avocado derived compound with demonstrated bioactivity against many diseases. It exists in different compartments of the avocado fruit; including the seed, which is typically discarded. To valorize this waste stream, a sustainable method to extract AVO in large quantities was developed. The AVO-rich extracts were prepared by solvent-based extraction followed by saponification. The amount of AVO in enriched extracts were measured by LC/MS after each modification of extraction and saponification parameters. The developed method obtained promising results in both small and large scale, which helps to increase the yields for industrial applications. Cytotoxicity of these AVO-rich extracts in acute myeloid leukemia cells was confirmed. To complete the method, the AVO-rich extract was purified by consuming the minimum amounts of reagents needed for purification. This developed method represents a simple, accurate and feasible method to extract AVO from avocado seed.