With an effort to understand the influence of permeation characteristics of hyperosmotic agents on tissue clearing progress and to look for effective concentrations that minimise the side effect for clinical applications, porcine stomach tissues (mucosa) applied with a mixed solution of glycerol and dimethyl sulfoxide (DMSO) are investigated with near-infrared reflectance spectroscopy and optical coherence tomography. Four solutions of 80% glycerol, 50% DMSO, 50% glycerol with 20% DMSO (GD1) and 30% DMSO (GD2) are studied, respectively. The significant improvement in light transmittance and thus enhancement of light penetration through tissue are demonstrated for all solutions. The development and effect of optical clearing accomplished by the four solutions in an increasing order for 50% DMSO, 80% glycerol, GD1, and GD2 are corresponding well to the rate and degree of water loss induced by the four solutions, which are in the same increasing order.