Biofilm structure and functionality depend on extracellular polymeric substances (EPS), but analytical methods for EPS often lack specificity which limits progress of biofilm research. EPS were extracted from aerobic granular sludge and analyzed with frequently applied colorimetric methods. The colorimetric methods were evaluated based on their applicability for EPS analysis. EPS fractions of interest were proteins, sugars, uronic acids and phenolic compounds. The applied methods (Lowry method, bicinchoninic acid assay, phenol sulfuric acid method, carbazole sulfuric acid method) were investigated in terms of their sensitivity towards the selected standard compound. Interference of compounds present in EPS with the colorimetric methods was further evaluated. All methods showed to be highly depending on the choice of standard compound and susceptible towards interference by compounds present in EPS. This study shows that currently used colorimetric methods are not capable of accurately characterizing EPS. More advanced methods are needed to be able to draw conclusions about biofilm composition, structure and functionality. Display omitted •Colorimetric EPS analysis is very sensitive towards the standard selection.•Analysis of single EPS compounds is prone to interference by other EPS compounds.•EPS quantification with colorimetric methods can easily give inaccurate results.•New approaches and methods are needed to accurately characterize EPS.