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We report the quantitative, label-free analysis of protein--protein interactions in free solution within picoliter volumes using backscatter interferometry (BSI). Changes in the refractive index are measured for solutions introduced on a PDMS microchip allowing determination of forward and reverse rate constants for two-mode binding. Time-dependent BSI traces are directly fit using a global analysis approach to characterize the interaction of the small heat-shock protein [alpha]-Crystallin with two substrates: destabilized mutants of T4 lysozyme and the in vivo target [beta]B1-Crystallin. The results recapitulate the selectivity of [alpha]B-Crystallin differentially binding T4L mutants according to their free energies of unfolding. Furthermore, we demonstrate that an [alpha]A-Crystallin mutant linked to hereditary cataract has activated binding to [beta]B1-Crystallin. Binding isotherms obtained from steady-state values of the BSI signal yielded meaningful dissociation constants and establishes BSI as a novel tool for the rapid identification of molecular partners using exceedingly small sample quantities under physiological conditions. This work demonstrates that BSI can be extended to screen libraries of disease-related mutants to quantify changes in affinity and/or kinetics of binding.