The performance of bioanalytic systems used for biowarfare agent detection, food or water toxin analysis relies on quantifiable and repeatable biomolecular interactions. Microsphere-based systems have come to be recognized as the platform of choice for next generation detection technologies. Microsphere-based immunoassay design is critically dependent on the complex interplay between buffer and bead properties as well as operational parameters. In previous work, we successfully developed physics-based computational tools for modeling sample detection using microspheres in microfluidic environments [1,2]. The models fully integrate Lagrangian transport of the beads, convective-diffusive transport of analyte and biomolecular surface binding reactions on the beads. Building of this multiphysics simulation framework, we present a methodology for optimization of microsphere-based assay design in this paper. The methodology is demonstrated via parameterization of a prototype sensor platform.