Membrane bioreactor fouling is a complex process, which is typically driven by extracellular polymeric substances (EPS), a complex mixture of polysaccharides, proteins, lipids, humic substances, and other intercellular polymers. While much is known about fouling in aerobic membrane reactors, far less is known about fouling in anaerobic membrane bioreactors (AnMBR). Much of this knowledge, including EPS extraction methods, has been extrapolated from aerobic processes and is commonly assumed to be comparable. Therefore, several extraction methods commonly used for aerobic EPS quantification, including ultrasonication, ethylenediaminetetraacetic acid (EDTA), and formaldehyde plus sodium hydroxide (CH2O+NaOH), were evaluated to determine the most suitable extraction method for EPS of anaerobic microorganisms in an AnMBR. To maximize EPS yields, each extraction was performed four times. Experimental results showed that the EDTA method was best for EPS quantification, based on chemical oxygen demand (COD), dissolved organic carbon (DOC), and protein yields: 1.43 mg COD/mg volatile suspended solids (VSS), 0.14 mg DOC/mg VSS, and 0.11 mg proteins/mg VSS. In comparison, the CH2O+NaOH method maximized the extraction of carbohydrates (0.12 mg carbohydrates/mg VSS). However, multiple extraction cycles with EDTA and ultrasonication exhibited lower extracellular adenosine triphosphate (ATP) concentrations compared to CH2O+NaOH extractions, indicating lower levels of released intracellular substances. Successive EPS extractions over four cycles are better able to quantify EPS from anaerobic microorganisms, since a single extraction may not accurately reflect the true levels of EPS contents in AnMBRs, and possibly in other anaerobic processes.