To achieve economic viability, biorefineries need to increase efficiency through characterization of byproducts for the purpose of valorization. One such byproduct is the liquid stream produced after autohydrolysis pretreatment, autohydrolyzate liquor, which contains valuable organic derivatives of hemicellulose and lignin from biomass. To characterize the autohydrolysis liquor, we employed a novel method for such liquor analysis that uses electrospray ionization and ion dopants in combination with tandem mass spectrometry using a quadrupole-time-of-flight mass spectrometer. Electrospray expands current analysis of such liquors through softer ionization. Ion dopants provide for differentiation of the complex mixture components without requiring derivatization or preliminary separation. The dopants-ammonium chloride and sodium hydroxide-primarily target and enhance ionization of hemicellulosic or lignin derivative species, respectively, based on the species' differing functionalities. Valuable structural information can be gleaned from these enhanced species by ion isolation and collision-activated dissociation (CAD), which reveals the presence of hemicellulosic or lignin derivative functionalities. These ionization techniques coupled with CAD enabled us to not only confirm the presence of low molecular weight ions, such as vanillin, as previously seen with gas chromatography-mass spectrometry but also expand the characterization to high molecular weight species. This expanded knowledge of the composition of autohydrolyzate liquor opens up the potential to develop lucrative co-products from this stream in a commercial biorefinery. (C) 2016 Elsevier Ltd. All rights reserved.