Standard methods for glucose quantification during high-solids enzymatic hydrolysis often neglect changes in hydrolysate density, leading to significant analytical errors and unrealistic process yields (>100%). To address this critical flaw, this research develops and validates a density-corrected calculation method ( C G ) while quantifying the error associated with the common, apparent approach ( C G 1 ). Theoretical modeling predicted a substantial overestimation by the uncorrected method, a finding confirmed during the 40% solids hydrolysis of extruded corn. Experimentally, the uncorrected ( C G 1 ) method progressively overestimated the true glucose concentration, with the discrepancy reaching approximately 15% after 24 hours. This resulted in a significantly inflated apparent glucose yield of 106.5% compared to the plausible 92.9% yield calculated with the corrected ( C G ) method. The results demonstrate that neglecting density is a major source of inaccuracy in high-solids analysis. Therefore, incorporating measured hydrolysate density via the validated ( C G ) method is essential for reliable monitoring, optimization, and comparison of high-solids bioprocesses for biofuel and biochemical production.