A new method involving the non-covalent binding of Coomassie Brilliant Blue G-250 (CBBG) to alkali-soluble lignin was developed. The binding of the dye to alkali-soluble lignin caused an increase in visible absorption at the maximum wavelength of 630 nm or 640 nm. A linear correlation of the absorbance at their maximum absorbing peak with alkali-soluble lignin concentration was observed. Lignin estimation in black liquor showed that the result of the new method and the gravimetric methods after acidification were closer to quantitative information than that obtained from UV spectroscopy. The isothermal titration calorimetric experiments, and Fourier Transform Infrared (FTIR) spectroscopy comparative analysis of precipitates washed by water, 4% ethanol, and 95% ethanol indicated that CBBG was bound to alkali-soluble lignin, and the binding was non-covalent. This potential method is reproducible, rapid, and cheap, and there is little or no inference from carbohydrate degradation products.