We present a method for characterizing the adsorption of solutes in microfluidic devices that is sensitive to both long-lived and transient adsorption and can be applied to a variety of realistic device materials, designs, fabrication methods, and operational parameters. We have characterized the adsorption of two highly adsorbing molecules (FITC-labeled bovine serum albumin (BSA) and rhodamine B) and compared these results to two low adsorbing species of similar molecular weights (FITC-labeled dextran and fluorescein). We have also validated our method by demonstrating that two well-known non-fouling strategies [deposition of the polyethylene oxide (PEO)-like surface coating created by radio-frequency glow discharge plasma deposition (RF-GDPD) of tetraethylene glycol dimethyl ether (tetraglyme, CH(3)O(CH(2)CH(2)O)(4)CH(3)), and blocking with unlabeled BSA] eliminate the characteristic BSA adsorption behavior observed otherwise.