We have recently proposed a computer-based method utilizing a thresholding algorithm (the Otsu method) to provide a convenient way of measuring the cell-free layer width in vivo and in vitro. However, this method does not seem to be a universal method that can be applied to all microvascular studies. Thus, we examined four different histogram-based thresholding algorithms (Otsu, intermode, minimum and second peak) to provide a technical suggestion on the selection of a suitable thresholding algorithm for the cell-free layer measurement. All the measurements were taken in microvascular flows in the rat cremaster muscle recorded with a high-speed camera. The width of the cell-free layer manually measured was compared with that determined by the automated method utilizing the four thresholding algorithms. With our experimental system, results showed that the cell-free layer width determined by the minimum algorithm was in best accordance with the manual measurement. We concluded that the accuracy of the automated methods for determination of the cell-free layer width would depend on the image quality, in particular on the contrast between the red blood cell core and background, which might differ due to the different microscopic setup. Therefore, one may need to examine several appropriate thresholding methods when selecting the best suitable algorithm for the experimental conditions.