The iFOLD® Protein Refolding System 2 is a detergent-free kit to screen for optimal refolding conditions for proteins found in inclusion bodies. The kit provides a systematic evaluation of 95 conditions representing different buffering systems, pH values, and combinations of salt, redox agent, and refolding additives. The system consists of inclusion body purification reagents and a pre-dispensed 96-well plate-based matrix of refolding buffers. Following cell lysis, membrane components and contaminating proteins trapped within inclusion body pellets are removed by a series of washes with a non-detergent sulfobetaine (NDSB). The purified inclusion bodies are reduced and denatured by addition of Tris(2-carboxyethyl) phosphine (TCEP) and guanidine hydrochloride and refolded by rapid dilution into the iFOLD System 2 detergent-free 96-well buffer matrix. Each kit is sufficient for screening up to 96 refolding conditions (95 experimental and 1 control well) for a single protein.