The microbial adhesion to hydrocarbons (MATH) test is one of the most common method to determine the hydrophobicity of cell surfaces. Despite its prevalence, no standard test parameters are used in literature, making a direct comparison of data almost impossible. Criticism also focuses on test parameters that may mask hydrophobic interactions and hence lead to erroneous test results. We methodically investigated the impact of different MATH test parameters on the calculation of the cell surface hydrophobicity of Streptococcus thermophilus, a widespread exopolysaccharide-producing lactic acid bacterium used in the production of fermented milk products. Besides composition and ionic strength of the buffer used for cell re-suspension, we observed a pronounced time dependency of the turbidity of the cell suspension during phase separation due to sedimentation and/or cell lysis. A new modification of the MATH assay was applied to enable the determination of cell surface hydrophobicity of long chain-forming bacteria. As the cell surface hydrophobicity was not altered during exponential growth phase, we assume that the cell surface and its capsular exopolysaccharide layer are not changed during cultivation.