To better assess the potential use of bio-oil as a chemical platform and fuel, a gas chromatography/mass spectrometry (GC/MS) technique is applied to characterize qualitatively and quantitatively a pine wood bio-oil produced by fast pyrolysis. An external calibration method using three different internal standards (toluene, 1,1,3,3-tetramethoxypropane, and 1-octanol) is performed to quantify 10 target compounds of bio-oil from different chemical families [2-propen-1-ol, 2-butanone, acetic acid, furfural, 2(5H)furanone, 2,5-dimethyoxy-tetrahydrofuran, 3-methyl-1,2-cyclopentanedione, 2-methoxy-4-propyl, vanilline, and levoglucosan], which were previously selected from the qualitative analysis of the bio-oil composition. To evaluate the reliability of this method using internal standards or without them, an exhaustive precision study is performed assessing the instrumental, intraday, and interday precisions. This study results in an overall good reproducibility of the method using any internal standard or without them. Finally, a comparative assessment of the different internal standards using statistical criteria (one-way analysis of variance and relative standard deviation) is carried out, showing that any of them can be used for bio-oil quantification. Thus, this study shows the analytical method suitability for the quantification of some bio-oil compounds by GC/MS.