ABSTRACT The North American overdose crisis remains at epidemic levels, with over 1 million lives lost over the past decade. This crisis is primarily driven by the emergence of high- potency opioids such as fentanyl, in unregulated (“street”) drug markets. However, the volatility of these markets has also led to the emergence of other even more potent opioids such as carfentanil and nitazene-class opioids, along with adulterants such as benzodiazepines. These emerging drug threats produce complex overdose presentations and difficulties in overdose reversal, all of which contribute to overdose mortality. Drug checking services, which provide individuals with analytic information regarding the presence of compounds in drugs of unknown composition, are increasingly employed in North America to prevent overdose mortality during the era of high-potency synthetic opioid contamination. However, current efforts to implement and expand access to drug checking services are hampered, primarily because current available technologies have logistical, technical, and cost-related barriers that impede their feasibility as point-of-care interventions. This is exacerbated by the need for ongoing assay development to ensure that drug checking technologies can continue to accurately detect and identify novel high-potency drug threats in North American drug markets. Meeting these needs is critical to advance the effectiveness of drug checking at the population level, particularly for drug checking service providers, individuals who use drugs, and medical examiners undertaking forensic investigations of overdose mortality. The aim of the current project is therefore to develop and validate a novel point-of-care technology, known as DoseCheck, for rapid non-targeted identification of emerging drug threats. Specifically, we will: 1) validate the DoseCheck system’s capacity to differentiate between and within classes of opioid, stimulant, depressant, and anesthetic compounds in non-targeted analyses of unregulated drugs; 2) undertake timed exercises to evaluate the capacity of our team to rapidly adapt the DoseCheck system to detect and differentiate a novel emerging drug threat; and 3) test the capacity of the DoseCheck system to identify and differentiate distinct opioid metabolites within human biomatrices. Achieving these aims will generate high impact and highly translational findings that will support the expansion of adaptive, analytically sophisticated, and low-cost point-of-care drug checking services and thereby contribute to a reduction in overdose mortality.