Sialic acids are biosynthesized by almost all organisms as a 9-carbon carboxylated monosaccharide and are integral components of glycoconjugates. More than 40 naturally occurring sialic acid derivatives of the three main forms of sialic acids, the N-acetyl- and N-glycolylneuraminic acid and 2-keto-3-deoxy-nonulosonic acid have been identified. Due to the great importance of sialic acids as key mediators in a plethora of cellular events, including cell-cell recognition and cell-matrix interactions, their analysis in biologic samples is useful for a deeper understanding of the various (patho)physiological processes and of value in disease diagnosis and monitoring. In this review we summarize the methodology developed to isolate and liberate sialic acids from biologic samples as well as the chromatographic, electromigration and hyphenated techniques available for their separation and analysis. A critical evaluation of the biological relevance of the results obtained by analyzing sialic acids in biologic samples is also presented.