BACKGROUND: Dried blood spot (DBS) sampling is a simple, cost-effective, and minimally invasive alternative to venipuncture for measuring exposure biomarkers in public health and epidemiological research. DBS sampling provides advantages in field-based studies conducted in low-resource settings and in studies involving infants and children. In addition, DBS samples are routinely collected from newborns after birth (i.e., newborn dried blood spots, NDBS), with many states in the United States permitting access to archived NDBS samples for research purposes. OBJECTIVES: We review the state of the science for analyzing exposure biomarkers in DBS samples, both archived and newly collected, and provide guidance on sample collection, storage, and blood volume requirements associated with individual DBS assays. We discuss recent progress regarding analytical methods, analytical sensitivity, and specificity, sample volume requirements, contamination considerations, estimating extracted blood volumes, assessing stability and analyte recovery, and hematocrit effects. METHODS: A systematic search of PubMed (MEDLINE), Embase (Elsevier), and CINAHL (EBSCO) was conducted in March 2022. DBS method development and application studies were divided into three main chemical classes: environmental tobacco smoke, trace elements (including lead, mercury, cadmium, and arsenic), and industrial chemicals (including endocrine-disrupting chemicals and persistent organic pollutants). DBS method development and validation studies were scored on key quality-control and performance parameters by two members of the review team. RESULTS: Our search identified 47 published reports related to measuring environmental exposure biomarkers in human DBS samples. A total of 28 reports (37 total studies) were on methods development and validation and 19 reports were primarily the application of previously developed DBS assays. High-performing DBS methods have been developed, validated, and applied for detecting environmental exposures to tobacco smoke, trace elements, and several important endocrine-disrupting chemicals and persistent organic pollutants. Additional work is needed for measuring cadmium, arsenic, inorganic mercury, and bisphenol A in DBS and NDBS samples. SIGNIFICANCE: We present an inventory and critical review of available assays for measuring environmental exposure biomarkers in DBS and NDBS samples to help facilitate this sampling medium as an emerging tool for public health (e.g., screening programs, temporal biomonitoring) and environmental epidemiology (e.g., field-based studies).