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Colorimetric determination method of D-galacturonic acid in reverse micellar solution has been investigated in order to assay pectinase catalyzed enzymatic hydrolysis of pectin in reverse micellar solution as a model study for bio-scouring of cotton in organic media. D-galacturonic acid produced by the hydrolysis of pectin could be detected by a specific condensation reaction with naphthoresorcinol. Condensation products showed clear blue color in reverse micellar solution that can be monitored by a spectroscopic determination method. Apparent rate of hydrolysis of pectin with pectinase enzyme in the reverse micellar solution obtained by the spectroscopic determination method was found to be slower than that in aqueous media, whereas the apparent rate of removal of the pectin from cotton fabrics by the pectinase assisted bio-scouring process in reverse micellar system was appreciably faster than that in aqueous media. The above difference between the results from the model study and from the practical bio-souring study would be attributed to the influence of cotton wax exist on actual cotton substrate that acts as gluing material for pectin and inhibit hydrolysis of pectic substances. Quick removal of cotton wax must be taken into account in the bio-scouring process to obtain effective scouring results.