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We have developed a high throughput technology for the functional discovery and validation of drug targets associated with viability of cancer cells.A second generation of Tet-regulated Functionally Validated bar-coded lentiviral pooled 30K shRNA library designed for the set of 10, 000 druggable and cell signaling genes was developed by validation of more than 100, 000 shRNA constructs.An optimal shRNA cassette design was developed by screening over 60 different shRNA cassettes that contain combinations of the most widely used structural features.The FV shRNA library is transduced into a cell line, the cell fraction with a specific phenotype is selected (e.g.by FACS), and functional shRNAs and corresponding target genes are identified by high throughput sequence analysis.Data will be presented that demonstrate the application, problems, and solutions for genome-wide functional analysis with arrayed and pooled shRNA libraries for several cancer cell models.We will present the following: 1) Comparison of pooled and array formats, 2) Lessons and solutions for RNAi screens, 3) High throughput validation of functional shRNAs, 4) Optimization of the shRNA expression cassette, and 5) Examples of negative and positive RNAi screens for cancer models.