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Abstract Aims There is a critical need for better biomarkers so that heart failure can be diagnosed at an earlier stage and with greater accuracy. The purpose of this study was to design a robust mass spectrometry (MS)‐based assay for the simultaneous measurement of a panel of 35 candidate protein biomarkers of heart failure, in blood. The overall aim was to evaluate the potential clinical utility of this biomarker panel for prediction of heart failure in a cohort of 500 patients. Methods and results Multiple reaction monitoring (MRM) MS assays were designed with Skyline and Spectrum Mill PeptideSelector software and developed using nanoflow reverse phase C18 chromatographic Chip Cube‐based separation, coupled to a 6460 triple quadrupole mass spectrometer. Optimized MRM assays were applied, in a sample‐blinded manner, to serum samples from a cohort of 500 patients with heart failure and non‐heart failure (non‐HF) controls who had cardiovascular risk factors. Both heart failure with reduced ejection fraction (HFrEF) patients and heart failure with preserved ejection fraction (HFpEF) patients were included in the study. Peptides for the Apolipoprotein AI (APOA1) protein were the most significantly differentially expressed between non‐HF and heart failure patients (P = 0.013 and P = 0.046). Four proteins were significantly differentially expressed between non‐HF and the specific subtypes of HF (HFrEF and HFpEF); Leucine‐rich‐alpha‐2‐glycoprotein (LRG1, P