The current article presents a mathematical description of the distribution of the enzyme reaction product around an enzyme-linked immunosorbent assay (ELISA) region of finite length created within a microfluidic channel. An analytical expression has been derived for this diffusion-reaction system relating the measured signal to the various operating parameters in the limit of slow diffusion across the assay segments. The predictions of this model have been shown to agree well with the recent experimental reports by Yanagisawa et al. on such a device. The current analysis also shows that quantitating ELISAs based on measurements made in the interfacial region between two assay segments is prone to error. However, such errors can be practically eliminated if the assay signal is collected from a region where this quantity deviates from its asymptotic limit far away from the interface by less than 5%. Moreover, the mathematical analysis suggests that the axial extent of an assay region (L) in these devices may be reduced to about 3 mm before signal cross-talk with its neighboring segments begins to affect the quantitation process. The reported value for L corresponds to a sample volume requirement of 3 nL per assayed analyte in a 10 μm deep and 100 μm wide microfluidic channel, which is nearly 3 orders of magnitude smaller than that required on microarray based platforms.