Recombinant protein overexpression of large proteins in bacteria often results in insoluble and misfolded proteins directed to inclusion bodies. We report the application of shear stress in micrometer-wide, thin fluid films to refold boiled hen egg white lysozyme, recombinant hen egg white lysozyme, and recombinant caveolin-1. Furthermore, the approach allowed refolding of the much larger protein, cAMP-dependent protein kinase A (PKA). The reported methods require only minutes, which is >100-times faster than conventional, overnight dialysis. This rapid refolding technique could significantly shorten the times, lower costs, and reduce the waste streams associated with protein expression for a wide-range of industrial and research applications.