A model analyte, the M13 virus, was detected through the change in the Brownian motion of a population of microparticles. Epi-fluorescence microscopy was used to simultaneously track antibody-coated and bare microparticles to unambiguously measure the diffusion coefficient and demonstrate multiplexed detection. The sensitivity of the diffusometry assay was high enough that individual virus-to-particle binding ratios could be detected. Analysis of the experimental errors indicated that the primary limitation in the sensitivity of this technique was the variation in the size of the population of microparticles. Analysis of the diffusion measurement results indicated that the change in the drag coefficient of the virus-particle assembly was not a simple sum of the drag coefficients of the individual components and the rate of particle-particle reaction was slower than would be predicted from the uncoupled particle hydrodynamics. The possibility of using diffusometry for sensing and proteomics applications is examined.