Photosystem II subunit S (PsbS) is a membrane protein that plays an exclusive role in non-photochemical quenching for photoprotection of plants under high-light conditions. The activation mechanism of PsbS and its pH-induced conformational changes are currently unknown. For structural investigation of PsbS, effective synthesis of PsbS with selective isotope or electron-spin labels or non-natural amino acids incorporated would be a great asset. Here we present cell-free (CF) expression as a successful method for in vitro production of PsbS that would allow such incorporation. The addition of several detergents, liposomes and lipid nanodiscs was tested for achieving soluble CF expression of PsbS. We have optimized the CF method to yield soluble PsbS of ∼500 ng/μl using a continuous-exchange method at 30 °C, along with a successful purification and refolding of PsbS in n-Dodecyl β-D-maltoside (β-DM) detergent. We expect that the presented protocols are transferrable for in vitro expression of other membrane proteins of the Light-Harvesting Complex family.