This chapter presents an in-depth exploration of biological matrices in forensic science, highlighting their roles, characteristics, and forensic significance. Conventional matrices such as blood and urine remain the backbone of forensic toxicology due to their ability to provide precise, real-time information on toxic substances and drug use. Blood offers unparalleled insight into physiological impairment, while urine is widely used for its non-invasive collection and extended detection window. Beyond these, alternative matrices including hair, nails, sweat, saliva, breast milk, and meconium broaden the scope of forensic analysis by enabling detection over longer or specialized timeframes. These matrices are particularly valuable when conventional specimens are unavailable, degraded, or compromised. Postmortem and veterinary matrices such as vitreous humor, liver, kidney, and bone extend applications into medico-legal and animal-doping contexts, providing unique stability and retrospective exposure data. Advances in analytical technologies, particularly high-resolution mass spectrometry and micro-sampling methods, have revolutionized sensitivity, speed, and scope across these diverse matrices. Each biological specimen presents specific advantages and challenges in terms of collection, stability, and interpretive accuracy, demanding tailored protocols to ensure reliability. Importantly, their forensic applications extend beyond criminal and postmortem toxicology into anti-doping programs, where matrices like blood, urine, and hair underpin the detection of prohibited substances and maintenance of sporting integrity. As a whole, this chapter underscores the scientific, legal, and ethical significance of biological matrices, emphasizing their critical role in strengthening the evidentiary foundation of forensic investigations and anti-doping controls. Future developments in omics technologies and biosensor platforms promise to enhance interpretive power, expand detection panels, and establish biological matrices as indispensable tools for justice, safety, and fair competition.