In this chapter, the discussion centers on the critical framework of prohibited substances and methods within global sports governance, highlighting their significance in safeguarding athlete health, maintaining fair play, and preserving the integrity of competition. The historical trajectory of anti-doping regulation is traced from early Olympic concerns in the 1960s to the establishment of the WADA in 1999 and the harmonization of international rules under the 2004 World Anti-Doping Code. At the heart of this framework lies the annually updated Prohibited List, which categorizes substances and methods into distinct sections based on their physiological impact, performance-enhancing potential, health risks, and ethical implications. This chapter explains how different classes of substances, such as anabolic agents, peptide hormones, stimulants, narcotics, and masking agents, can artificially enhance performance while exposing athletes to serious medical risks ranging from cardiovascular disorders to psychological complications. Beyond chemicals, prohibited methods including blood manipulation, chemical tampering, and gene or cell doping are examined as evolving threats to fairness and health. The discussion also highlights the complexities introduced by therapeutic use exemptions (TUEs) and the ongoing struggle to detect designer drugs crafted to evade testing. By analyzing the rationale, mechanisms, and enforcement strategies surrounding doping, this chapter underscores the broader ethical dimension: the need to protect the spirit of sport as a celebration of natural talent, discipline, and human potential. As science advances, anti-doping regulation must remain vigilant, adaptive, and globally coordinated. Ultimately, this chapter frames prohibition not as restriction alone, but as a collective safeguard ensuring that competitive sports remain an authentic and inspiring human endeavor.