A rapid, sensitive and accurate response of analytical techniques to resolve health issues, such as low limit identification in special biological environment (i.e. complex mixtures of proteins) remains to being a key aspect in modern proteomics. In fact, extensive efforts to obtain sensitivity enhancements with detection limits even down to the single molecule have been devoted in the nanotechnology framework. Recently, innovative approaches based on the properties of colloidal nanoparticle (NP) assemblies have led to the development of novel diagnostic methods with sensitivity enhancements for single-molecule protein monitoring/ identification/detection. Among them, surface-enhanced Raman spectroscopy (SERS) benefits from its higher detectable response to binding of a single protein (sensitivity) and also, very importantly, from its multiplexing capabilities due to the narrow nature of detected peaks from Raman reporter molecules. Since SERS retains the fingerprinting capabilities of Raman spectra, the internal modes of a reporter molecule brought at metallic NPs junctions, where strong field enhancement occurs, can be used as diagnostic tools. Specific attention has been given to SERS-based immunoassays. Indeed, the combination of the high sensitivity provided by SERS and the strong binding specificity of antibody-protein ensures that SERS-based detection platform are suitable tools for biomedical and biochemical analysis, clinical diagnosis and biosensor.