Four methods for measuring diffusion coefficients were compared on a microfabricated fluidic device using rhodamine 6G as the analyte. The measurements were made using a static imaging method and three dynamic methods-stopped flow, varying the applied potential (E-field method), and varying the detection length (length method). Under conditions where analyte-wall interactions (adsorption) are minimized, e.g. in a 50/50 (v/v) methanol/aqueous buffer, the stopped flow (2.71+/-0.09x10(-6) cm(2) s(-1)), E-field (2.684+/-0.005x10(-6) cm(2) s(-1)) and the static imaging (2.69+/-0.02x10(-6) cm(2) s(-1)) measurements were all within experimental error of one another and previously reported values. Under 100% aqueous conditions, however, the diffusion coefficient measured dynamically was 11% larger than that measured statically. Diffusion coefficients for rhodamine B, fluorescein, 2',7'dichloro-fluorescein (DCF), rhodamine 6G, tetramethylrhodamine labeled glutamic acid and isoleucine, and fluorescein conjugated bovine serum albumin and ovalbumin were also measured using the static imaging method.