ABSTRACT Hepatitis C, the inflammatory liver disease caused by hepatitis C virus (HCV), claims approximately 400,000 lives each year from secondary hepatocellular carcinoma and cirrhosis-induced liver failure. Direct-acting antiviral agents can cure > 95% of the estimated 71 million cases of hepatitis C found globally, yet because of underdiagnosis, only 7% of patients are currently receiving treatment. The diagnostic testing algorithm for HCV necessitates multiple clinic visits. Individuals in vulnerable populations often lack the means to meet this high follow-up burden. Increasing the prevalence of HCV treatment to reflect the 95% cure rate requires simplification of diagnostic testing. To this end we will develop a sample-to-answer HCV viral load (VL) test that meets or exceeds the minimal acceptable criteria as defined by the hepatitis C global stakeholder consortium. To achieve this goal, we will combine Redbud Labs’ expertise in microfluidics and systems with the diagnostic assay development expertise of the Diagnostics Program at PATH (Seattle, WA). In Aim 1 Redbud Labs will optimize each assay step to the microfluidic environment using Redbud technologies and commercially available reagents. In Aim 2 PATH will design assay primers and probes. In Aim 3 Redbud Labs will validate the assay by performing it end-to-end using a breadboard system. Redbud technologies are production-ready, which will minimize the risk of degradation in assay performance as we move through cartridge integration in future Phase II work.