Understanding the depth of penetration of near-infrared (NIR) light in biological tissue is critical for enhancing clinical applications of near-infrared spectroscopy (NIRS). The current knowledge of NIRS penetration depth primarily stems from mathematical models, numerical simulations, and phantom studies, with a notable knowledge gap derived from real animal models. By sequentially obstructing light from traversing in a porcine kidney tissue model, we derived the depth distribution of NIR light experimentally and better characterized its dependence on the distance between the light source and photodetector. We collected four replicates of data from six different source-detector distances (SDSs) and found that both the maximum and mean depths of penetration of NIRS increase with the SDS. Linear relationships can be derived between the SDS and the maximum depth, and the square root of the SDS and the mean depth.