Dry Blood Spots (DBS) have been used in combination with liquid chromatography-mass spectrometry for targeted proteomics to identify sensitive and specific novel biomarkers. DBS presents several advantages over other traditional blood sampling methods. This review discusses the past, present and future of the technology, focusing on studies with clinical and population relevance. Arguments for and against DBS are presented by discussing technological advances, particularly those related to Mass Spectrometry (MS) and Multiple Reaction Monitoring (MRM), sample preparation issues, disease biomarkers, pharmacokinetics, and pharmacodynamics. There will be a focus on proteomic studies that rely on DBS as a sampling method. In this context, numerous studies on the diagnosis and treatment of several diseases. To date, proteomic reports of studies using DBS have shown that DBS can facilitate diagnosis and prognosis. DBS offers several advantages that make it a viable option for many fields. Moreover, some of its disadvantages can be easily overcome through automation to increase reproducibility and reduce protocol variability and standardization of parameters such as the volume of sample used. Within this context, here we propose to review the advantages and disadvantages of using DBS for blood proteomics and provide an understanding of how current DBS-based protocols are being conducted for future standardization and protocol optimization.