The incorporation of unnatural amino acids (uAA) can introduce novel functional groups into proteins site-specifically, with important applications in basic sciences and protein engineering. However, uAA incorporation can impact protein expression and functional activity depending on its location within the proteina process that is not yet completely understood and difficult to predict. Therefore, practical applications often necessitate a time-consuming optimization of uAA location by individual gene cloning, expressions, purification, and evaluations for each location tested. To address this limitation, we introduce a streamlined and versatile in vitro system to rapidly express and screen uAA-containing proteins without cumbersome cell culturing or purification procedures. We utilized this technology to simultaneously screen 24 different t4-lysozyme mutants with different uAA incorporation sites in a matter of hours, compared to weeks-long workflow of conventional methods. Screening data offered a mechanistic explanation to some effects of uAA incorporation on expression and activity. Despite these insights, rational prediction of such effects remained challenging, further confirming the value of a rapid screening approach. Biotechnol. Bioeng. 2017;114: 2412-2417. (c) 2017 Wiley Periodicals, Inc.