Docosanol is the only US Food and Drug Administration (FDA) approved over-the-counter topical product for treating recurrent oral-facial herpes simplex labialis. Validated analytical methods for docosanol are required to demonstrate the bioequivalence of docosanol topical products. A gas chromatography/selected ion monitoring mode mass spectrometry (GC/SIM-MS) method was developed and validated for docosanol determination in biological samples. Docosanol and isopropyl palmitate (internal standard) were separated on a high-polarity GC capillary column with (88% cyanopropy)aryl-polysiloxane employed as the stationary phase. The ions of 83 and 256 were selected to monitor docosanol and isopropyl palmitate, respectively; the total run time was 20 min. The GC/SIM-MS method was validated in accordance with US FDA guidelines, and the results met the US FDA acceptance criteria. The docosanol calibration standards were linear in the 100-10000 ng/mL concentration range ( >0.994). The recoveries for docosanol from the receptor fluid and skin homogenates were >93.2% and >95.8%, respectively. The validated method was successfully applied to analyze ex vivo human cadaver skin permeation samples. On applying Abreva® cream tube and Abreva® cream pump, the amount of docosanol that penetrated human cadaver skin at 48 h was 21.5 ± 7.01 and 24.0 ± 6.95 ng/mg, respectively. Accordingly, we concluded that the validated GC/SIM-MS was sensitive, specific, and suitable for quantifying docosanol as a quality control tool. This method can be used for routine analysis as a cost-effective alternative to other techniques.