A non-aqueous reversed phase high performance liquid chromatographic (NARP-HPLC) method for determination of coenzyme Q10 in pharmaceutical preparations has been developed using Kromosil C8 column with acetonitrile and isopropyl alcohol (84:16, v/v) as a mobile phase. Photodiode array (PDA) detector set at 210 nm was used for monitoring of the eluents. The method is simple, rapid, selective and capable of separating all process impurities at trace level with detection limits <0.1 μg/ml. It has been validated with respect to accuracy, precision, linearity, and limits of detection and quantification. The linearity range was 50–300 μg/ml. The percentage recoveries ranged from 95.10 to 101.02. The method was found to be suitable not only for monitoring the reactions during the process development but also quality assurance of coenzyme Q10. For identification of related substances atmospheric pressure chemical ionisation-mass spectrometry (APCI-MS) was used.