In vitro and ex vivo studies on drug metabolism and stability are vital for drug development and pre-clinical safety assessment. Traditional in vitro models, such as liver enzyme (S9) fractions and microsomes, often fail to account for individual variability. Personalized models, including 3D cell models and organoids, offer promising alternatives but may not fully replicate physiological processes, especially for Cytochrome P450 (CYP) families involved in extrahepatic metabolism. A major challenge in these studies is the low stability and expression of CYP enzymes. This study aimed to stabilize native CYP activity in vitro by developing an optimized buffer formulation. Initial experiments using recombinant CYP supersomes and liver microsomes identified 45 μM cysteine, 4 mM dithiothreitol (DTT), and 300 μM phosphocholine (PC) as the most effective stabilizers. The applicability of these stabilizers was subsequently confirmed in primary human brain tissue, where they enabled the successful determination of CYP2D6 activity. This highlights the stabilizing buffer's utility for enhancing CYP functionality in diverse tissue types, including the brain, which plays a critical role in cerebral detoxification and drug metabolism. These findings suggest that specific enzyme stabilization can enable comprehensive evaluations of CYP function in ex vivo tissue samples, advancing the development of organoid human tissue models and supporting drug metabolism research.