Emerging advancements in protein biotherapeutics are driving the need for more sensitive and accurate bioanalytical methods for their quantification. Serving as an orthogonal technology to the traditional ligand binding assays (LBAs), LC-MS has been routinely adopted for quantitative measurement of protein levels in bioanalytical laboratories. While various MS approaches have been investigated by researchers, bottom-up workflows using targeted LC-MRM strategy remain the most common. Here, to leverage selectivity, accurate mass spectrometry was used to quantify a series of signature peptides in rat plasma. The novel QTOF mass spectrometer involves improvements in MS/MS sampling efficiency with the use of a Zeno trap which enables greater sensitivity for quantification. In addition, an overview of the fundamental steps to develop a highly sensitive and selective quantitative assay for signature peptides will be presented.