2-methoxyestradiol (2ME) is an endogenous 17β-oestradiol metabolite that exerts antiproliferative, antiangiogenic and antitumour activity on cancer cells both in vitro and in vivo. However, the use of 2ME as a potential anticancer agent is limited due to its poor oral bioavailability coupled to a short elimination half-life. In an attempt to improve the oral bioavailability and expand the drug targets, three sulphamoylated 2ME analogues were designed using in silico modelling and subsequently synthesized. A screening limit of 5 μg/ml for each analogue using a liquid chromatography tandem mass spectrometer (LC-MS/MS) method for 2ME analogues in serum and solvent was established. Therapeutically relevant oral bioavailability was assessed for these 2ME analogues using a murine oral absorption model (CD-1 mice) where the presence of these synthetic analogues in serum samples was assessed at two hours post dosing at 150 mg/kg of individual compounds. Blood was collected and analysed for the presence of the dosed compound and potential metabolites via LC-MS/MS. Results indicated that these analogues were present in serum above the screening limit at two hours post dosing and that there is merit to further investigation into the mode and mechanism(s) of action of ESE-15-one and ESE-15-ol and ESE-16 in vivo.