Abstract: An immunoassay of proteolytic protein fragments is described, including immunoassays of proteolytic fragments of Insulin-like Growth Factor Binding Proteins (IGFBPs). In one embodiment, a sandwich-type “two-site” immunoassay involves two different recognition antibody partners, in which one antibody is specific for a proteolytic epitope of a protein fragment, and the other is specific for the protein fragment. An assay embodiment involves a first-step capturing of the protein fragment with a specific anti-protein antibody that binds to the proteolytic epitope of the protein fragment, and a second-step detection of the bound protein fragment with an antibody directed against the protein fragment. The various embodiments of the systems and methods of the invention are exemplified by immunoassays for proteolytic fragments of Insulin-like growth factor binding proteins (IGFBPs), such as IGFBP-1, IGFBP-3, and IGFBP-5.