Advances in software and high resolution, high mass accuracy mass spectrometers have expanded their functionality beyond traditional data dependent acquisition (DDA) methods. Using a single platform, an orthogonal quadrupole time-of flight (QqTOF) mass spectrometer, the TripleTOF 5600, we have investigated the feasibility of implementing large-scale targeted quantitative assays, derived from discovery type data sets, using scheduled, high resolution multiple reaction monitoring (sMRM-HR) mass spectrometry. We assessed the selectivity and reproducibility of MRM-HR, also referred to as parallel reaction monitoring (PRM), measuring standard peptide concentration curves as well as system suitability assays. We specifically compared the robustness and accuracy of MRM-HR assays to traditional SRM workflows on triple quadrupole instruments. To determine the utility of sMRM-HR for large-scale targeted quantitative assays, we retention time scheduled over 500 peptides in a single LC-MS acquisition. High resolution and high mass accuracy of the full scan MS/MS spectra resulted in sufficient selectivity to monitor numerous MS/MS fragment ions per analyte precursor and provided flexibility for post-acquisition assay refinement and optimization. To demonstrate its applicability to biological samples, whole cell lysates from several E. coli wild-type and mutant strains were quantitatively assayed by sMRM-HR to confirm a previously generated candidate list of differentially expressed proteins. The ease of developing and implementing sMRM-HR assays derived directly from DDA discovery workflows on the same high resolution instrument platform facilitates downstream validation studies targeting many peptides for MS/MS level quantitation. This work provides a robust MRM-HR workflow for rapidly moving from discovery analysis to large-scale, multiplexed, targeted quantitation.