Image thresholding is a widely used technology for a lot of computer vision applications, and among various global thresholding algorithms, Otsu-based approaches are very popular due to their simplicity and effectiveness. While the usage of Otsu-based thresholding methods is well discussed, the performance analyses of these methods are rather limited. In this paper, we first review nine Otsu-based approaches and categorize them based on their objective functions, preprocessing, and postprocessing strategies. Second, we conduct several experiments to analyze the model characteristics using different scene parameters both on synthetic images and real-world cell images. We put more attention to examine the variance of foreground object and the effect of the distance between mean values of foreground and background. Third, we explore the robustness of algorithms by introducing two typical kinds of noises under different intensities and compare the running time of each method. Experimental results show that NVE, WOV, and Xing’s methods are more robust to the distance of mean values of foreground and background. The large foreground variance will cause a larger threshold value. Experiments on cell images show that foreground miss detection becomes serious when the intensities of foreground pixels change drastically. We conclude that almost all algorithms are significantly affected by Salt&Pepper and Gaussian noises. Interestingly, we find that ME increases almost linearly with the intensity of Salt&Pepper noise. In terms of algorithms’ time cost, methods with no preprocessing and postprocessing steps have more advantages. All these findings can serve as a guideline for image thresholding when using Otsu-based thresholding approaches.