Tight junction (TJ) modulation is a promising approach for improving drug bioavailability by enhancing the absorption of active pharmaceutical ingredients. However, the application of many different test methods to determine the efficacy of new TJ modulators (TJMs) or to assess different compounds is accompanied by a lack of comparable results, reducing the rational evaluation and commercial marketing of these pharmaceutical excipients. The establishment of unified testing methods can fill this gap and offers the opportunity to compare results from different laboratories. Furthermore, the calculation of a TJ modulation score allows the objective comparison of TJ modulators and facilitates the selection of appropriate candidates. In this study, eight well-known TJ modulators were tested with a focus on four different in vitro bioassays carried out with MDCK cells. The extent of TJ modulation was determined by transepithelial electric resistance (TEER) measurements and permeability studies with mannitol. To evaluate tolerability, cell viability (MTT) and cytotoxicity (CellTox Green) assays were performed, and TEER regeneration was monitored for 24 h after exposure. With the exception of labradimil, seven TJ modulators caused significant TEER reduction of up to 100 %. For five compounds, an enhancement of mannitol permeation was observed. As expected, first-generation enhancers exhibited lower cell compatibility than mechanism-based modulators. Based on the experimental results of this study, for the first time, an evaluation system (tight junction modulator scoring system, TJMSS) is presented that provides a ranking of the tested modulators depending on weighted parameters. Such a system offers the possibility of rational formulation development for drugs requiring improved absorption.