Purpose of review Optimized platelet isolation is essential for preserving platelet integrity and function. This review elucidates the scientific rationale behind common workflows, explores modern strategies for their refinement, and aims to improve practices in translational hematology. Recent findings Recent research has increasingly highlighted the multifaceted roles of platelets beyond their traditional function in hemostasis, underscoring the necessity for precise and reliable isolation techniques. Inadequate or improper isolation methods can inadvertently trigger platelet activation. Although a wide array of isolation protocols exists, their selection is often based on empirical convention rather than scientifically validated criteria. This review provides a critical evaluation of commonly employed platelet isolation methods, examining their advantages, limitations, and the challenges they pose, particularly with respect to minimizing unintended platelet activation. In addition, we discuss innovative approaches that aim to overcome these limitations, including the application of supramolecular anticoagulants. Finally, we explore the transformative potential of artificial intelligence driven technologies to optimize or even replace conventional platelet isolation techniques. Summary This review analyzes established platelet isolation protocols, clarifying their rationale and key overlooked factors. It explores the potential of emerging technologies, particularly artificial intelligence, to transform platelet research, raising the question of whether these advancements could replace traditional methods.