The ever-increasing number of sequenced genomes and subsequent sequence-based analysis has provided tremendous insight into cellular processes; however, the ability to experimentally manipulate this genomic information in the laboratory requires the development of new high-throughput methods. To translate this genomic information into information on protein function, molecular and cell biological techniques are required. One strategy to gain insight into protein function is to observe where each specific protein is subcellularly localized. We have developed a pipeline of methods that allows rapid, efficient, and scalable gene cloning, imaging, and image analysis. This work focuses on a high-throughput screen of the Caulobacter crescentus proteome to identify proteins with unique subcellular localization patterns. The cloning, imaging, and image analysis techniques described here are applicable to any organism of interest.