This review discusses various sample pretreatment methods for the analytical assessment of endocannabinoids in biological fluids and tissues. Techniques like liquid-liquid extraction, protein precipitation, SALLE, SPME, and micro-solid phase extraction (µ-SPE) are investigated. The findings show that SALLE and SPME provide phase separation as a fast and environmentally friendly method and allow high sensitivity in the analyses. The analysis of endocannabinoids, particularly, 2-AG, is challenging due to its tendency to isomerize into 1-AG and its lack of stability. These challenges are highlighted, emphasizing the need for optimized analytical methods to ensure accurate and reliable results. In conclusion, this study demonstrates the applicability of effective extraction techniques as an alternative to traditional methods to obtain reliable results in endocannabinoid analyses and provides a new perspective to the literature.