Measurement of low concentrations of estrogens, encountered in pre-pubertal children, men, and postmenopausal women, is important for numerous clinical applications. We describe a method for high sensitivity analysis of estrogens that uses two-dimensional chromatographic separation and tandem mass spectrometry detection. Aliquots of serum or plasma samples are combined with stable isotope-labeled internal standard and estrogens are extracted with methyl t-butyl ether. The solvent is evaporated, estrogens derivatized to form dansyl derivatives, and the samples are analyzed. Quantitation is performed using triple quadrupole mass spectrometer equipped with electrospray ion source using positive ion mode ionization and multiple reaction monitoring acquisition.