β-agonists are prohibited substances for athletes according to the World Anti-Doping Agency and are banned as growth promoters for livestock in most countries. The illicit use of β-agonists is a significant concern for both human and animal health. Hair analysis serves as a valuable material for monitoring the abuse of β-agonists. This manuscript aims to provide a comprehensive overview of the application of hair analysis in monitoring β-agonist abuse. The morphology, structures, and composition of hair are summarized, along with an introduction to the mechanisms of β-agonist incorporation into hair. Considering various factors, the treatment procedures for β-agonist detection in hair, with special attention to the authentic matrix, are outlined. Additionally, diverse screening and confirmatory methods are reviewed. Particular emphasis is placed on novel purification processes and innovative analytical technologies employed for the analysis of β-agonists in hair.