Recent progress in the study of cytokinin metabolism in plants indicates that quantitative analysis of cytokinin nucleotides is essential for elucidation of early steps of the biosynthetic pathway. However, traditional procedures for purification and quantification of cytokinin cannot discriminate the various nucleotides. We describe here a method for separation and determination of cytokinin nucleotides through a series of anion-exchange column chromatography steps followed by liquid chromatography-mass spectrometry. This method enabled us to analyze the amount of each species of cytokinin nucleotide in plant tissues.