Objectives: To evaluate a new method for assessing the radio-opacity of endodontic sealers and to compare radio-opacity values with a well-established standard method. Methods: The sealers evaluated in this study were AH Plus ® (Dentsply DeTrey GmbH, Konstanz, Germany), Endo CPM Sealer (EGEO SRL, Buenos Aires, Argentina) and MTA Fillapex ® (Angelus Dental Products Industry S/A, Londrina, Parana, Brazil). Two methods were used to evaluate radio-opacity: (D) standard discs and (S) a tissue simulator. For (D), ten standard discs were prepared for each sealer and were radiographed using Digora ® phosphor storage plates (Soredex; Orion Corporation, Helsinki, Finland), alongside an aluminium stepwedge. For (S), polyethylene tubes filled with sealer (n 5 10 for each) were radiographed inside the simulator as described. The digital images were analysed using Adobe Photoshop ® software v. 10.0 (Adobe Systems, San Jose, CA). To compare the radio-opacity among the sealers, the data were analysed by ANOVA and Tukey's test, and to compare methods, they were analysed by the Mann-Whitney U test. To compare the data obtained from dentin and sealers in method (S), Student's paired t-test was used (50.05). Results: In both methods, the sealers showed significant differences, according to the following decreasing order: AH Plus, MTA Fillapex and Endo CPM. In (D), MTA Fillapex and Endo CPM showed less radio-opacity than aluminium. For all of the materials, the radio-opacity was higher in (S) than in (D). Compared with dentin, all of the materials were more radio-opaque. Conclusions: The comparison of the two assessment methods for sealer radio-opacity testing validated the use of a tissue simulator block. Dentomaxillofacial Radiology (2015Radiology ( ) 44, 20140422. doi: 10.1259 Cite this article as: Malka VB, Hochscheidt GL, Larentis NL, Grecca FS, Fontanella VRC, Kopper PMP. A new in vitro method to evaluate radio-opacity of endodontic sealers. Dentomaxillofac Radiol 2015; 44: 20140422.