Proteomics offers a deeper understanding of the protein events influencing cellular and biological function. However, identifying key regulated proteins and their implicated signaling pathways represents a major analytical challenge to researchers. This is due to the broad dynamic range of protein abundance in biological samples and the inherent limitations of the instrumentation and methodologies utilized to probe and detect minute target proteins in complex cellular environments. In this webinar, we discuss targeted mass spectrometry (MS)-based approaches and the various strategies designed to reduce sample complexity through improved sample preparation and enrichment. Focusing on proteins involved in the AKT/mTOR signaling pathway, we provide guidance toward targeted MS assay development and validation, including robustness, precision, and accuracy of these assays. Finally, we’ll examine results of multiplexed IP to targeted MS assays compared to existing immunoassay techniques.