A series of dried blood spot (DBS) detection methods for doping agents have been developed in the last two decades. The DBS technique minimizes invasiveness and reduces storage and shipping costs. Recently, the World Anti-Doping Agency announced the use of DBS for the 2022 Beijing Winter Olympic Games and Paralympic Games owing to the advantages of the DBS application in routine doping control. Therefore the further development of detection methods for doping agents in DBS is important and urgent. This review summarizes five aspects of DBS application in doping analysis: sample collection, storage conditions, pretreatment, instrumentation and validation according to the Prohibited List issued by the World Anti-Doping Agency, and proposes some suggestions for future studies of DBS in doping analysis.