The degree of substitution for sulfobutyl ether b-cyclodextrin (SBE-b-CD) has historically been characterized by nuclear magnetic resonance spectroscopy, but this approach provides gross values for the degree of substitution. The capillary electrophoresis method described here resolves the mixture of positional and regional isomers based on the degree of sulfo butyl ether substitution. Indirect UV detection has been used, where the detection of cyclodextrin is accomplished by a decrease in background absorbance of the background electrolyte buffer benzoic acid. Ten peaks of SBE-b-CD were well resolved with an optimum pH of 8.0 using an Agilent 50 μm id, 56 cm length bare fused silica capillary. The system suitability requirements were passed as per United States Pharmacopeia (USP), and acceptence criteria for averge degree of substitution 6.2 to 6.9 were met.