Coenzyme Q₁₀ (Q₁₀) plays an important role in mammals for energy production in the mitochondria, and as a potent antioxidant. Oxidation ratio (% oxidized in relation to total Q₁₀) has been proposed as an important biomarker. A sensitive and reproducible HPLC-ECD method was developed for determination of reduced and oxidized Q₁₀ in canine plasma and heart tissue. Chromatographic separation was achieved in 10 min using a Waters Nova-pak C₁₈ column and a mobile phase with lithium perchlorate in ethanol/methanol/2-propanol. The validation showed satisfying results. Excellent linear correlation was found (r² > 0.9997), intra-and inter-day precisions were below 6.5% (n = 5) and recoveries were between 89 and 109% (n = 5). Sensitivity stated as Lower Limit of Quantification (LLOQ) was 10 nM. Acceptable stability of both extracted and un-extracted samples was observed. The plasma concentration range of total Q₁₀ was found to be between 0.64 and 1.24 µg/mL. Comparison with a developed LC-MS/MS method showed a correlation of r = 0.85 for reduced Q₁₀ and r = 0.60 for oxidized Q₁₀ (N = 17). However, average results were around 30% lower for ubiquinol using the LC-MS/MS method as compared with the HPLC-ECD analysis. The two methods are therefore not considered to be interchangeable.