The use of alternative matrices such as oral fluid and hair has increased in the past decades because of advances in analytical technology. However, there are still many issues that need to be resolved. Standardized protocols of sample pretreatment are needed to link the detected concentrations to final conclusions. The development of suitable proficiency testing schemes is required. Finally, interpretation issues such as link to effect, adulteration, detection markers and thresholds will hamper the vast use of these matrices. Today, several niche areas apply these matrices with success, such as drugs and driving for oral fluid and drug-facilitated crimes for hair. Once those issues are resolved, the number of applications will markedly grow in the future.