Holographic molecular binding assays detect target molecules binding to the surfaces of specifically functionalized probe beads by measuring the associated increase in bead diameter with holographic video microscopy. Holograms of individual colloidal beads are analyzed by fitting to analytic predictions of the Lorenz-Mie theory of light scattering, yielding measurements of bead diameter with the nanometer precision required to detect binding events. Holographic binding assays share the specificity and robustness of industry-standard bead-based assays. Direct holographic readout eliminates the processing time, expense and uncertainty associated with fluorescent labeling. The underlying technology for holographic particle characterization also has a host of other applications in biopharmaceuticals, semiconductor processing and fundamental research.