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BACKGROUND: 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) is a zwitterionic detergent commonly utilized in protein biochemistry-based applications. This study demonstrates for the first time the successful phase separation of CHAPS via salt addition. Elucidation of consolute solution temperature behavior and determination of binodal curve and tie-line length (TLL) enabled the subsequent development of CHAPS-based phase separation systems. The systems were then evaluated using a low-molecular-weight dye, Coomassie Brilliant Blue (CBB), and integral membrane protein, cytochrome P450 (CYP) 2C19. RESULTS: The anionic identity of salts was found to be critical, with sulfate and phosphate salts being successful in inducing phase separation of CHAPS at 4 ∘ C. CHAPS and (NH 4 ) 2 SO 4 mixtures were later found to have an upper consolute solution temperature (UCST). Binodal data were collected and fitted to an equation to allow determination of the minimum amount of CHAPS and (NH 4 ) 2 SO 4 required for the formation of phase separation systems. In CHAPS/(NH 4 ) 2 SO 4 systems, CBB was found to partition extensively to the micelle-rich phase (MRP) with an extraction yield of 83 ± 0.32%, while CYP 2C19 was successfully recovered with an extraction yield of 46 ± 7.58%. CONCLUSION: Phase separation of CHAPS achieved at low temperature via sulfate salt addition can be exploited for the extraction of hydrophobic molecules. The outcomes of this study propound the scope for application of CHAPS as a component of phase separation systems. TLL determinationTLLs for a series of phase separation systems with different mass compositions (Table 2) were determined. The systems were formed by mixing pre-determined amounts of CHAPS and (NH 4 ) 2 SO 4 . Each mixture was incubated at 4 ∘ C and vortexed