To make clear the generation mechanism of microbending loss in optical fibers and effectively estimate and test microbending loss, this paper analyzed two mechanisms, i.e., microbending caused by pure bending and that by coupling, briefly described the most relevant and effective theoretical formulas, and pointed out the most important factors affecting the microbending loss. Two methods to test microbending loss were proposed for the first time, respectively using an optical fiber reel covered by metal mesh and a double-slot plate. In light of the transverse strain and pure bending theory, two corresponding simulation algorithms were established respectively. Depending on the decomposition and simulation of microdistortion and microbending of the tested optical fibers in test platforms, the microbending loss was estimated. The established platforms were then used for testing. The verification results show that the calculated values of the proposed simulation algorithms are in good agreement with the measured results, and the proposed test methods can be employed for standard formulation and extended.