Hydrothermal liquefaction (HTL) is an efficient second-generation technology for conversion of biomass or biomass waste products into bio-oils for e.g. the transport sector. The product consists of bio-oil of varying viscosity and composition, as well is aqueous and gas-phase byproducts. In order to guide Optimization of the HTL process with regard to conditions, catalysts; and feedstock, there is a need for standardized methods for product analysis. We have developed analytical methods to investigate the chemical composition of bio-oils produced in a continuous HTL plant using 11 different types of feedstocks and model compounds (sucrose, cellulose, sunflower oil, casein, gluten, cellulose mixed with protein, manures, sewage sludge, and Dried Distillers Grains with Solubles, DDGS). Bio-oils aqueous phase byproducts were analyzed directly by gas-chromatography (GC) coupled with mass spectrometry (MS), while fatty acids (C10-C18) were specifically quantified, using GC (with flame ionization detection) after derivatization into their methyl esters. In addition we developed and applied a new high-performance liquid chromatography time-of-flight mass spectrometry (HPLC-TOF-MS) method for detection Of fatty acids (C14-C22) in the aqueous phase byproduct. Generally HTL bio-oils had a. complex composition showing 100-200 separate peaks in the GC-MS chromatograms, but for most feedstocks, 30-50 peaks accounted for a large fraction of the total peak area. Grouping of identified products into chemical families provided a useful overview for characterization of the bio-oils. Fatty acids (C16 and C18) were major products from HTL of protein (gluten), sunflower, oil, DOGS, sewage sludge, and some manure, feedstocks, while other important compound groups in bio-oils included phenols, cyclopentenes, indoles, pyrroles, and amides. The aqueous phase contained products such as cyclopentenones, pyrazines, and fatty acids, also found in the bio-oils, which were probably present as micelles suspended in the aqueous phase.