© 2014 American Chemical Society. The interrogation of highly diverse repertoires of heterogeneous cell populations on a single cell basis increases the likelihood that a cell with unique characteristics will be identified. We have developed a new single cell analysis system comprising millions of bundled subnanoliter volume bioincubation chambers for the identification and recovery of target specific antibody secreting cells (ASCs). This platform integrates dual surface screening with dedicated user driven data analysis and automated cell recovery enabling multiple biophysical parameters to be tracked for millions of antibody leads in parallel. This direct clone analysis and selection technology is a clear deviation from current microfabricated well-based approaches and offers drastically enhanced screening throughput, simultaneous dual surface analysis, and rapid automated single cell recovery. The technology is also applicable to screening both bacterial and mammalian antibody secreting cells. We demonstrate the implementation and feasibility of this platform in identifying target specific antibodies from bacterial, hybridoma, and B cell libraries.