In this chapter, a comprehensive overview of extraction techniques employed in forensic toxicology and anti-doping analysis is presented. Accurate detection of drugs, metabolites, and prohibited substances from complex biological matrices such as blood, urine, hair, and oral fluid depends largely on the effectiveness of sample preparation. The chapter systematically discusses conventional and modern extraction techniques, highlighting their principles, advantages, limitations, and applications in both forensic casework and sports drug testing. LLE remains a widely applied classical approach due to its simplicity and cost-effectiveness, yet it often requires large solvent volumes and may lack selectivity. Solid-phase extraction (SPE) offers improved cleanup and reproducibility, while solid-phase microextraction (SPME) introduces solvent-free sampling with minimal sample handling. More advanced miniaturized techniques, such as dispersive liquid–liquid microextraction (DLLME), combine high enrichment factors with reduced solvent consumption. Similarly, stir bar sorptive extraction (SBSE) enhances analyte recovery for trace-level detection, particularly in environmental and biological applications. Emerging methods like supported liquid extraction (SLE) and magnetic solid-phase extraction (MSPE) demonstrate significant progress in automation, reusability, and environmental sustainability, offering higher sensitivity for complex toxicological and doping analyses. The dilute-and-inject technique, although simple, is limited to clean matrices and often requires high-performance instruments to tolerate direct sample loading. By comparing the strengths and drawbacks of each method, this chapter emphasizes that no single technique is universally superior. Instead, method selection depends on analyte properties, matrix complexity, available resources, and the analytical goal. Together, these extraction strategies form the foundation of reliable toxicological interpretation and fair enforcement in anti-doping practices, ensuring scientific accuracy and ethical integrity.