Abstract HPLC–UV and GC/MS determination of aldehydes in bio-oil were evaluated. HPLC–UV preceded by derivatization with 2,4-dinitrophenylhydrazine allows separation and detection of bio-oil aldehydes, but the derivatization affected the bio-oil stability reducing their quantitative applicability. GC/MS determination of aldehydes was reached by derivatization with o-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine hydrochloride. Two approaches for this reaction were evaluated. The first: “in solution derivatization and head space extraction” and the second: “on fiber derivatization SPME”, the latter through an automatic procedure. Both sample treatments allows the quantification of most important aliphatic aldehydes in bio-oil, being the SPME approach more efficient. The aldehyde concentrations in bio-oil were ∼2% formaldehyde, ∼0.1% acetaldehyde and ∼0.05% propionaldehyde.