Procedures were developed for analysis and purification of phosphorylated isoprenoids by reversed-phase HPLC. Dimethylallyl diphosphate (C5), geranyl diphosphate (C10), farnesyl diphosphate (C15), geranylgeranyl diphosphate (C20), and farnesylgeranyl diphosphate (C25) were cleanly separated on a C18 column by gradient elution with acetonitrile and 25 mM NH4HCO3. Reversed-phase HPLC was used to directly analyze extracts for phosphorylated isoprenoids and to follow the progress of enzyme-catalyzed reactions without having to hydrolyze the phosphate esters before analysis. Examples are provided for geranylgeranyl diphosphate synthase, geranylgeranylglyceryl phosphate synthase, and squalene synthase. The separations were run on preparative scales to purify products from the enzymatic reactions for mass spectrometry and to purify synthetic substrates.