The extensive metabolism and treatment of low doses of estrone (E1), estradiol (E2) and estriol (E3) in preclinical animal species necessitates a sensitive analytical method to identify or quantify the estrogens in biological matrixes. In this study, a highly sensitive and specific method based on the derivatization of E1, E2 and E3 with 10-ethyl-acridine-2-sulfonyl chloride (EASC) coupled with liquid chromatography-ion-trap mass spectrometry with APCI-MS (MRM) identification of estrogens has been developed. The EASC derivatization of E1, E2 and E3 introduces an acridine functional group into estrogen molecules. The carbonyl group in EASC core results in the formation of a phenoxide negative ion by the intramolecular keto-enol isomerization that can be accepted a [H]+ and readily ionized in commonly used LC mobile phases. Derivatives are sufficiently stable to be efficiently analyzed by LC-APCI-MS and show an intense protonated molecular ion at m/z [M+H]+ in positive-ion mode. The collision-induced dissociation of molecular ion forms a distinctive product ion at m/z 222.6, corresponding to the protonated 10-ethyl-acridine moiety. The selected reaction monitoring, based on the m/z [M+H]+ → m/z 222.6 transitions, is highly specific for estrogen derivatives. Therefore, the facile EASC derivatization coupling with LC-APCI-MS analysis allows the development of a highly sensitive and specific method for the identification of trace levels of estrogens in urine of root vole (Microtus oeconomus Pallas).