Black cumin seed (Nigella sativa L.) is a widely used spice and herb, where thymoquinone (2-isopropyl-5-methyl-1,4-benzoquinone) is the major bioactive compound. Here, a stable isotope dilution (SID) gas chromatography-mass spectrometry (GC-MS) technique was developed for the quantification of thymoquinone. A doubly deuterated thymoquinone ([(2)H2]-thymoquinone) was synthesized for the first time with more than 93% deuteration degree shown by mass spectrometry and proton nuclear magnetic resonance ((1)H NMR). This compound was used as an internal standard for the quantification of thymoquinone using a SID GC-MS method. The validation experiment showed a recovery rate of 99.1 ± 1.1% relative standard deviation (RSD). Standard addition and external calibration methods have also been used to quantify thymoquinone, which cross-validated the developed stable isotope dilution assay (SIDA). In comparison to external calibration and standard addition methods, the SIDA method is robust and accurate. The concentration of thymoquinone in five marketed black cumin seed oils ranged between 3.34 and 10.8 mg/mL by use of SID GC-MS.