Quantification of endogenous biomarkers in clinical studies requires careful evaluation of a number of assay performance parameters. Comparisons of absolute values from several clinical studies can enable retrospective analyses further elucidating the biology of a given biomarker across various study populations. We characterized the performance of a highly multiplex bioanalytical method for quantification of phosphatidylinositols (PI). Hydrophilic interaction chromatography (HILIC) and multiple reaction monitoring (MRM) were employed for targeted multiplex quantification. Odd-chain PI species that are not normally present in human plasma were utilized as surrogate analytes (SA) to assess various assay performance parameters and establish a definitive dynamic linear range for PI lipids. To correct for batch effects, Systematic Error Removal using Random Forest (SERRF) normalization algorithm was employed and used to bridge raw values between two clinical studies, enabling quantitative comparison of their absolute values. A high throughput method was developed, qualified, transferred to an automation platform and applied to sample testing in two clinical trials in healthy volunteers (NCT03001297) and stable Coronary Artery Disease (CAD, NCT03351738) subjects. The method demonstrated acceptable precision and accuracy (±30%) over linear range of 1–1000 nM for SA and 8-fold dilutional linearity for endogenous PI. We determined that mean-adjusted average QC performed best for normalization using SERRF. The comparison of two studies revealed that healthy subject levels of PI are consistently higher across PI species compared to CAD subjects identifying a potential lipid biomarker to be explored in future studies.