Oestrogen quantification in serum is challenging as concentrations are low, especially in men and post-menopausal women. Additionally, oestrogens do not ionise readily and structure similarities can lead to cross-reactivity and reduced specificity, especially with immunoassay. Full characterisation of oestrogen metabolism and its role in disease progression has therefore not been fully investigated. We aimed to develop and validate a liquid chromatography mass spectrometry method which separates 11 oestrogens and demonstrate its utility for in-vitro experiments and biological samples. Mass spectrometry parameters were optimised for each oestrogen on a Waters Acquity UPLC chromatography system coupled to a Waters Xevo TQ-XS mass spectrometer and electrospray ionisation source. Oestrogens were subsequently combined and eight columns from Waters and Phenomenex were screened to optimise chromatographic separation using a methanol/water (both with 0.1% formic acid) elution system. The column which provided the most favourable chromatographic parameters, Phenomenex Kinetex F5 2.6 μm 50×2.1 mm, was further optimised via gradient and investigation into mobile phase additives to produce an 8 min method with baseline resolution of all analytes. LOQ was less than 0.5 ng/ml for oestrone, oestradiol, 2-methoxyoestradiol and 16-hydroxyoestrone, 1 ng/ml for 11b-OHoestradiol, and 2-methoxyoestrone, and 5 ng/ml for oestriol and the 2 and 4 hydroxylated oestrogens. Accuracy (%bias) and precision (%CV) were assessed at 3 levels of concentration (low 2 ng/ml, medium 20 ng/ml and high 200 ng/ml). Accuracy ranged from 1 to 19, −4 to 16, and −8 to 10% (low, medium and high), precision ranged from 8 to 19, 3 to 17 and 3 to 9% (low, medium and high) excluding the 2 and 4 hydroxylated oestrogens below LOQ. Further experiments are under-way to improve sensitivity and extraction efficiency. This novel method separated 11 structurally similar oestrogens in 8 min and can now be applied to oestrogen analysis in-vitro and in biological samples.