Interstitial flow (IF) across the aortic walls induces mechanical stress on smooth muscle cells, altering their function in response to the flow. Due to the heterogeneous structure of the aorta, IF velocity varies locally. We previously developed a method for measuring local IF velocity by directly visualizing the movement of a fluorescence dye driven by the fluid flow using fluorescence microscopy and quantifying the velocity based on a one-dimensional convection-diffusion equation (Fukui et al., Sci. Rep. 2022:12(1), 5381). However, the original method had low measurement reliability due to the widely scattered data points in kymograph-based analyses, leading to low correlation coefficients. This study aimed to refine the method for improved reliability. Several modifications were introduced, including replacing the forward difference scheme with a central difference scheme in the convection-diffusion equation, minimizing image drift, compressing the image size of kymograph, and optimizing the image analysis region. These modifications enhanced data fitting to a linear trend, leading to more accurate interstitial flow velocity measurements. When applied to the dataset from Fukui et al., the modified method significantly improved measurement reliability, yielding correlation coefficients above 0.90 and velocity values several times higher than those obtained before modification. The proposed approach provides a more reliable technique for quantifying IF velocity in the aortic wall for future biomechanical studies.