The molecular size of meningococcal polysaccharides is an important physicochemical parameter that is related to immunogenicity and efficacy. A simple method for size-exclusion chromatography was developed, optimized, and applied for safe and rapid fractionation of meningococcal polysaccharide AC vaccine. Pooling of the fractions collected from size-exclusion chromatography was investigated and evaluated, rather than analyzing each fraction separately, for determining the percentages of meningococcal polysaccharide A and C that were eluted before the distribution coefficient of 0.5. Pooling is preferred rather than analyzing each fraction individually, as it is easily handled, faster, simpler, less expensive, more accurate, safe, and applicable. The developed method was validated and successfully applied for the determination of meningococcal polysaccharide vaccine serotype A and C in quality-control and commercial samples.