We investigated the application of 1-alkylamines, as additives to the mobile phase, to a quantification method for ubiquinone-9 (CoQ 9 ) and ubiquinone-10 (CoQ 10 ) in rat thigh muscle and heart using liquid chromatography–tandem mass spectrometry (LC–MS/MS). In the optimization of the analytical method, we found that 1-alkylamines mixed with CoQ 9 and CoQ 10 in the turbo ion sprayed solution formed the 1-alkylammonium adduct molecules of these compounds during the ionization process and that the intensity of the adduct ions was considerably higher than that of the protonated molecules ([M+H] + ) of these compounds. Furthermore, we investigated a variety of 1-alkylamines in the mobile phase for LC–MS/MS analysis to select the most appropriate 1-alkylamine for higher sensitivities of CoQ 9 and CoQ 10 . After these examinations, we found that methylamine was the most suitable additive for the mobile phase, allowing a 12.5-fold gain in signal intensity in the full ion mass spectrum compared with that without methylamine. The internal standard (IS) used was ubiquinone-11 (CoQ 11 ) for each analyte. The analytes and IS were extracted with methanol from the tissue homogenates at neutral pH and were injected into an LC–MS/MS with a turbo ion spray interface. The calibration curves for CoQ 9 (5–500μg/g in thigh muscle and 50–10,000μg/g in heart) and CoQ 10 (1–500μg/g in thigh muscle and 10–10,000μg/g in heart) showed good linearity. The method was precise; the relative standard deviations of the method for rat thigh muscle were not more than 13.5 and 9.0% for CoQ 9 and CoQ 10 , respectively, and those for rat heart were not more than 6.7 and 5.4% for CoQ 9 and CoQ 10 , respectively. The accuracies of the method for both rat thigh muscle and heart were good, with the deviations between the nominal concentration and calculated concentration of CoQ 9 and CoQ 10 typically being within 12.3 and 4.3%, respectively. This method provided reliable concentration levels for CoQ 9 and CoQ 10 in rat thigh muscle and heart.