A novel strategy for construction of protein libraries called "SIMPLEX: single-molecule PCR-linked in vitro expression" is described. A pool of genes is prepared and thereafter extensively diluted to give one molecule of DNA per well. Each individual molecule is separately amplified by PCR (single-molecule PCR) yielding a one-well-one-gene PCR library. Subsequently, the PCR library is directly transformed into a protein library by means of in vitro-coupled transcription/translation in an array format. Individual proteins in the library can be screened for target functions directly without further purification. The generated protein library is compatible with various selection methods. The strategy provides high-throughput construction and screening of protein libraries, and suits automation.