The Semi-Continuous Activated Sludge (SCAS) test of the ASTM “Standard Test Method for Biodegradability of Alkylbenzene Sulfonates” (D2667-89) was modified and used to compare the biological removal of new carbohydrate-based surfactants (CBS). The SCAS test was modified in three ways: the units were miniaturized, natural sewage replaced synthetic sewage, and analysis for dissolved organic carbon (DOC) replaced methylene blue active substance as the analytical method. The modified test distinguished between compounds that underwent minimal to complete biological removal by establishing a percent removal at a “steady state” (typically reached within two weeks). The method is inexpensive, easy, and efficiently compares biological removal of surfactants or other water-soluble compounds. This study shows that random substitution of hydroxyl hydrogens on glucose inhibits biological removal. In contrast specific regio-substitution at the 3-O or 1-O positions did not inhibit biological removal.