Abstract: An assay device includes a colorimetric testing assembly including a detection area, a fluid inlet, and a microfluidic network including a first path extending to the detection area and a second path extending to the detection area. When a fluid (e.g., a buffer fluid or a combined buffer and sample solution) is provided to the fluid inlet, a first portion of the fluid rehydrates a first dried reagent (e.g., a dried enzyme label) disposed along the first path to produce a first rehydrated reagent and a second portion of the fluid rehydrates a second dried reagent (e.g., a dried substrate) to produce a second rehydrated reagent. The first rehydrated reagent and the second rehydrated reagent are then sequentially delivered to the detection area by capillary-driven flow to perform the assay.