This work presents a method for molecular weight cut off (MWCO) characterization of nanofiltration membranes, in a broad range of acidic and alkaline environments. Polyethylene glycols (PEG) have been identified as suitable marker molecules with sufficient chemical stability under the harsh conditions of interest. PEG molecular weight distributions have been analyzed using gel permeation chromatography (GPC). To allow quantitative GPC analysis, a protocol is presented to overcome the problem of an overlapping salt peak in the GPC elugram. The method is applied to a well-known commercial nanofiltration membrane (NF-270, DOW FILMTEC™) in the pH range 2–12. This membrane has similar MWCO (270 g mol−1) and permeance (10 L m−2 h−1 bar−1) in acid environment and at neutral conditions. At pH = 12 a reversible increase was observed for the MWCO (380 g mol−1) and the permeance (12 L m−2 h−1 bar−1). This demonstrates the added value of our method to observe the change of MWCO as a function of pH during nanofiltration at the relevant conditions.