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The lack of high-throughput approaches for expression and screening of large enzyme libraries remains a major bottleneck for current enzyme engineering efforts. To address this need, we have developed a high-throughput, fluorescence-based approach for rapid one-pot, microscale expression, and screening of industrial enzymes. In this chapter, we present the protocol for integration of cell-free protein expression with activity screening of enzymes in two formats: (1) a 96-well plate format and (2) a microscale-array format. Our one-pot method is ideally suited for rapid, first pass screening of enzymes and can also be used to perform detailed mechanistic analysis such as measurement of kinetics, determination of optimum temperature, and to study enzyme inhibition.