Steroid estrogens contained in wastewater discharge from sewage treatment plants and agricultural run-off can alter endocrine function in exposed wildlife at part per trillion (ng/L) levels. Detection and quantification of estrogens in the environment at these levels pose numerous analytical challenges. This study aimed to develop a reproducible, accurate, and simple method for 17β-estradiol (E2) quantification from environmental water samples. Extraction efficiencies of three solid phase extraction (SPE) cartridges; ENVI-Carb®, Oasis HLB Glass® and Oasis HLB Plus® were tested by trial extractions of 500 mL ultrapure water spiked to 1 ng/mL, 10 ng/mL and 100 ng/mL. The Oasis HLB Plus® cartridges yielded the best mean extraction efficiencies of 120.50 ± 8.02 %; 113.23 ± 9.77% and 105.2 ± 21.16% for 100 ng/mL, 10 ng/mL and 1 ng/mL ultrapure water spikes respectively. Further tests of extractions efficiencies for field water samples showed reduced mean extraction efficiencies of 80.6 ± 13.1 % for concentrations ranging from 40-15000 ng/L. This suggests the need for further clean-up of heavily loaded aqueous samples. Detection and quantification was by LC/MS using electrospray ionization (ESI) in negative ionization mode. Presence of precursor ion 271 (m/z) and product ion 183 (m/z) identified E2. A limit of quantification (LOQ) of 10 ng/L was established by finding the lowest point on the calibration curve whose signal-to-noise ratio was at least 3. Validation of the LOQ by analysis of 7 replicates of 40 ng/L field spikes by the SPE-LC/ESI/MS procedure yielded a mean recoveries of 81.98 ± 8.5%.