Background: Ovarian cancer is the most lethal gynecologic malignancy in women, and high-grade serous ovarian cancer (HGSOC) is the most common subtype. Currently, no clinical test has been approved by the FDA for the detection of ovarian cancer in the general population. This underscores the critical need for the development of a robust methodology combined with novel technology to detect diagnostic biomarkers for HGSOC in the sera of women. Targeted mass spectrometry (MS) can be used to identify and quantify specific peptides/proteins in complex biological samples with high accuracy, sensitivity, and reproducibility. In this study, we developed and conducted analytical validation of a multiplexed Tier 2 targeted MS parallel reaction monitoring (PRM) assay for the relative quantification of 23 ovarian cancer protein biomarker candidates in sera. Methods: To develop a PRM method for our target peptides, we followed nationally recognized consensus guidelines for validating fit-for-purpose Tier 2 targeted mass spectrometry (MS) assays. The endogenous target peptide concentrations were calculated using the calibration curves for each target peptide. Receiver operating characteristic (ROC) curves were analyzed to evaluate the diagnostic performance of the biomarker candidates. Results: We describe the development and analytical validation of a multiplexed Tier 2 targeted MS PRM assay to quantify ovarian cancer protein biomarker candidates in sera. Among the 64 peptides corresponding to 23 proteins in our PRM assay, 24 peptides corresponding to 16 proteins passed the assay validation acceptability criteria. Six of these peptides from insulin-like growth factor-binding protein 2 (IBP2), sex hormone-binding globulin (SHBG), and TIMP metalloproteinase inhibitor 1 (TIMP1) were quantified in sera from a cohort of 69 patients with early-stage HGSOC, late-stage HGSOC, benign ovarian conditions, and healthy (non-cancer) controls. IBP2 was identified as a diagnostic biomarker candidate based on its significantly increased abundance in the late-stage HGSOC patient sera compared to the healthy controls and patients with benign ovarian conditions. Conclusions: This study developed and validated a multiplexed targeted MS PRM assay for diagnostic biomarker analysis in HGSOC. To evaluate the clinical utility of IBP2 for HGSOC detection, further studies need to be performed using a larger patient cohort.