Carotenoids and tocopherols are health-functional phytochemicals that occur in a wide range of fruit and vegetable crops. These two classes of compounds are synthesized from a common precursor, geranyl-geranyl pyrophosphate, and are typically analyzed separately via high-performance liquid chromatography (HPLC) techniques. Because carotenoids and tocopherols are present in many edible horticultural crops, it would be advantageous to measure them simultaneously in plant tissues. Herein we report a one-pass reverse-phase HPLC method for extraction and analysis of carotenoids and tocopherols in carrot that can be extended to other high-moisture plant organs. Elution times ranged from 5 minutes for α-tocopherol to 24 minutes for β-carotene. This method improves the efficiency of analyzing these compounds by up to 50%, and should increase the efficiency of assessing carotenoid and tocopherol profiles in horticultural crops.