17beta-Estradiol (17beta-E2) is a steroid with pleiotropic actions. In addition to being a sexual hormone, it is also produced in the brain where it modulates the reproductive axis. It has been shown that 17beta-E2 also acts on synaptic plasticity and plays a role in neurological pathways and in neurodegenerative diseases. Assaying this steroid in the brain is thus interesting to improve our knowledge of 17beta-E2 effects in the brain. However, 17beta-E2 concentration in the central nervous system has been reported to be of a few nanograms per gram wet weight (nanomolar range concentration); therefore, its quantification requires both an efficient extraction process and a sensitive detection method. Herein is presented a derivatization-free procedure based on solid-phase extraction followed by LC-MS/MS analysis, targeted on 17beta-E2, its isomer17alpha-E2, and its metabolites estrone (E1) and estriol (E3). This extraction process allowed reaching 96% 17beta-E2 recovery from the mouse brain. Limit of detection (LOD) and limit of quantification (LOQ) values of 0.5 and 2.5 pmol mL(-1), respectively, were reached for both 17alpha-E2 and 17beta-E2. LOD values for E1 and E3 were 0.01 and 0.025 pmol mL(-1), respectively. The variation coefficients for intra- and inter-assays were 6 and 14%, respectively, for both estradiol forms. The method was applied to assess estrogen levels in the mouse brain and hippocampus after 17beta-E2 acute (subcutaneous injection) and chronic (drinking water) physiological administration. Total estrogen levels were determined after enzymatic deconjugation and compared to free estrogen levels. While 17alpha-E2 was not detected in biological samples, 17beta-E2 and metabolite measurements highlight a local biotransformation of estrogens after physiological administration via drinking water. Graphical abstract Method workflow: After oral or subcutaneous Estradiol administration, mouse brain or hippocampus was removed. Samples were homogenized and prepared according to a liquid-liquid extraction, followed by a solid-phase extraction. Then, LC-MS/MS was optimized to quantify 17ss-E2, its isomer17alpha-E2, its metabolites estrone (E1) and estriol (E3) and their conjugates.