The biomedical community has the imperative to develop reliable, clinically relevant and generalizable bioassays that can be used to accurately recognize those individuals with early-stage disease or those patients who will respond to therapy, with the ultimate aim of achieving individualized medicine. In recent years, increasingly sophisticated proteomic screening technologies have been introduced, providing the biomedical community with a valuable new approach for the systematic discovery and validation of novel diagnostic, prognostic and therapeutic tools. Nevertheless, the complexity of the cellular milieu wherein a variety of macromolecules interact in dynamic fashion, combined with the complex clinical manifestation of chronic pathologies and widespread diversity of patient populations, mean that universal biomarkers will not be easily developed. In this review, the five key challenges that must be surmounted in order to advance the clinical impact of this nascent field are described, and plausible solutions based on the authors' own ongoing proteomic profiling of cardiovascular disease is outlined.