Updated project metadata. Non–islet-cell tumor hypoglycemia (NICTH) is a rare paraneoplastic phenomenon seen in both dogs and humans. NICTH syndrome is derived from incompletely processed forms of insulin-like growth factor–II (IGF–II) by tumors, commonly named as big IGF–II. In the present study, a previously developed targeted PRM MS-based method for cats have been optimized and applied to simultaneously quantify the levels of IGF–I, IGF–II, and IGFBP–3, and for the first time, the levels of big IGF–II in dogs. This method allows the absolute quantification of these proteins using a mixture of QPrEST™ proteins previously designed for humans, reducing the variations due to the methodology, and the amount of serum needed for the analysis.