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Nonislet-cell tumor hypoglycemia (NICTH) is a rare paraneoplastic phenomenon well described in dogs and humans. Tumors associated with NICTH secrete incompletely processed forms of insulin-like growth factor-II (IGF-II), commonly named big IGF-II. These forms have increased bioavailability and interact with the insulin and IGF-I receptor causing hypoglycemia and growth-promoting effects. Immunoassays designed for human samples have been used to measure canine IGF-I and -II, but they possess some limitations. In addition, there are no validated methods for measurement of big IGF-II in dogs. In the present study, a targeted parallel reaction monitoring MS-based method previously developed for cats has been optimized and applied to simultaneously quantify the serum 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 IGF proteins using a mixture of QPrEST proteins previously designed for humans. The method possesses good linearity and repeatability and has been used to evaluate the IGF-system in a dog with NICTH syndrome. In this dog, the levels of big IGF-II decreased by 80% and the levels of IGF-I and IGFBP-3 increased approximately 20- and 4-times, respectively, after removal of the tumor.