We report a method for high-throughput, cost-efficient empirical discovery of optimal proteotypic peptides and fragment ions for targeted proteomics applications using in vitro-synthesized proteins. We demonstrate the approach using human transcription factors, which are typically difficult, low-abundance targets and empirically derived proteotypic peptides for 98% of the target proteins. We show that targeted proteomic assays developed using our approach facilitate robust in vivo quantification of human transcription factors.