Aim: This in vitro study evaluated the effectiveness of detecting the presence of second mesiobuccal (MB2) canal in the maxillary first molars, using magnification and illumination. Materials and Methods: Access cavities of fifty extracted human maxillary first molars were prepared, and the floor of the pulp chamber was then explored to locate the MB2 canal in five stages: Stage I (direct vision), Stage II (under ×2.5 magnifying loupes without light-emitting diode [LED] light), Stage III (under ×2.5 magnifying loupes with LED light), Stage IV (under operating microscope at ×5), and Stage V (under operating microscope at ×12.8). Statistical Analysis: The descriptive statistics were tabulated using the kappa statistic to check the association between the five groups. Results: The operating microscope at ×5 and ×12.8 gave a diagnostic accuracy of 100%, followed by magnifying loupes with LED light which gave a diagnostic accuracy of 90% in detecting the presence of MB2 canal. The use of magnifying loupes without LED light and plain eyesight gave a comparatively lesser diagnostic accuracy, i.e., 76% and 68%, respectively. Conclusion: The operating microscope with a sensitivity and diagnostic accuracy of 100% was most effective in the detection of MB2 canals followed by magnifying loupes with LED light.