The study of molecular interactions in biological fluids is important as a research tool to elucidate molecular and biological function, for discovering or designing molecules that have a desirable function, and for measuring or detecting analytes for clinical diagnostic purposes. Microfluidics is an emerging technology that has proven useful for studying and controlling molecular interactions with the potential advantages of reduced sample and reagent volumes, short reaction times, portable instrumentation, and high throughput. At microscale dimensions, diffusive transport of many biologically relevant molecules can cover large fractions of a fluid channel in a short time (seconds to minutes). We have exploited this feature to analyze molecular interactions based on changes in the diffusive transport of one of the reacting components. Here, we present a new assay configuration for studying molecular binding interactions and report new developments in the fabrication and use of hydrogels for diffusion-based analysis. We also overview system configurations and analysis techniques that have proven useful for studying molecular interactions of biological analytes and discuss their ability to operate using complex fluids such as blood.