Direct infusion-electrospray ionization (ESI)-Ion-Trap MS and ESI-Ion-Trap MS2 were used for direct analysis of bio-oil from forest residue and reference bio-oils from cellulose and hardwood lignin. It was found that the bio-oil concentration and mode of MS analysis are important parameters in obtaining reproducible and structurally informative data. In order to study sensitivity and selectivity with ESI-Ion-Trap MS, a selection of model compounds were studied with and without dopants. Dopants included NaCl, formic acid and NH4Cl in positive ion mode and NaOH and NH4Cl in negative ion mode. NH4Cl addition can be used to distinguish carbohydrate-derived products from other bio-oil components. NaOH and NaCl additives produced the highest peak intensities in negative ion mode as deprotonated adducts and in positive mode as sodiated adducts, respectively. ESI-MS2 was used successfully for confirmation of individual target ions such as levoglucosan and cellobiosan, as well for some structural products of lignin. Simple bio-oil fractionation into hydrophilic and hydrophobic components provided less complex and more interpretive ion spectra.