The Biology/Disease-driven (B/D) working groups of the Human Proteome Project are alliances of research groups aimed at developing or improving proteomic tools to support specific biological or disease-related research areas. Here, we describe the activities and progress to date of the B/D working group focused on protein aggregation diseases (PADs). PADs are characterized by the intra- or extracellular accumulation of aggregated proteins and include devastating diseases such as Parkinson’s and Alzheimer’s disease and systemic amyloidosis. The PAD B/D working group aims for the development of proteomic assays for the quantification of aggregation-prone proteins involved in PADs to support basic and clinical research on PADs. Because the proteins in PADs undergo aberrant conformational changes, a goal is to quantitatively resolve altered protein structures and aggregation states in complex biological specimens. We have developed protein-extraction protocols and a set of mass spectrometric (MS) methods that enable the detection and quantification of proteins involved in the systemic and localized amyloidosis and the probing of aberrant protein conformational transitions in cell and tissue extracts. In several studies, we have demonstrated the potential of MS-based proteomics approaches for specific and sensitive clinical diagnoses and for the subtyping of PADs. The developed methods have been detailed in both protocol papers and manuscripts describing applications to facilitate implementation by nonspecialized laboratories, and assay coordinates are shared through public repositories and databases. Clinicians actively involved in the PAD working group support the transfer to clinical practice of the developed methods, such as assays to quantify specific disease-related proteins and their fragments in biofluids and multiplexed MS-based methods for the diagnosis and typing of systemic amyloidosis. We believe that the increasing availability of tools to precisely measure proteins involved in PADs will positively impact research on the molecular bases of these diseases and support early disease diagnosis and a more-confident subtyping.