For the first time, matrix-assisted laser desorption/ionization (MALDI) method was successfully applied for identification of the concentration of individual polyethylene glycol (PEG) oligomer used in the complex mixture solutions used for evaluation of separation performance of nanofiltration membranes. As a result, it was possible to plot MWCO curve based on the filtration complex solution contained PEG oligomers with a molecular weight of up to 2000 g/mol. To eliminate the influence of porous support, the dense membranes made of two polymers of intrinsic microporosity, poly(1-trimethylsilyl-1-propyne) (PTMSP) and poly(4-methyl-l-pentene) (PMP), were selected in this study. It was found that the molecular weight cut-off (MWCO) parameter for PTMSP and PMP determined by using of linear PEG were as 1550 +/- 30 and 910 +/- 30 g/mol, respectively. Such noticeable difference in MWCO was explained by the fact that PMP has about 2 times lower fraction accessible volume comparing with PTMSP (FAV(PTMSP) = 30%, FAV(PMP) = 16%). It was shown that the branched PEGs, trimethylolpropane ethoxylates (TMPE), can overcome the problem of high flexbility of linear PEGs. For example, the corresponded MWCO values for PTMSP and PMP determined with TMPE were 910 +/- 30 and 820 +/- 30 g/mol, respectively.