A new and superior one-filtration method for the determination of the molecular weight cut-off (MWCO) of aqueous based nanofiltration and ultrafiltration membranes has been developed using the widest range of polyethylene glycol oligomers as MWCO probes of any MWCO method so far. This method was enabled by a new, high resolution oligomer separation and detection using high performance liquid chromatography (HPLC) coupled with an evaporative light scattering detector (ELSD). The refined method can determine the MWCO of membranes over a MW range from 678 to 4594 g mol(-1) with a molecular weight difference of just 44 g mol(-1) and a bonus further one point extension to 6000 g mol(-1) giving the widest range and most precise difference of MWs that can be resolved of any single filtration MWCO method that exists. MWCO determination of five commercial membranes from GE Osmonics (TM) and Millipore showed good agreement with manufacturer and literature values, confirming the accuracy of the method. As this new method has significant advantages over all other existing aqueous MWCO determinations (i.e. single filtration, higher resolution over a wider MW range, low cost MWCO molecular probes), it is suggested that it could be adopted as the new standard for determining aqueous MWCO over a MW range from 678 to 6000 g mol(-1).