Glycan profiling is a common strategy that is used to determine the distribution of N-linked glycans, O-linked glycans and glycolipid associated complex carbohydrate structures that are part of various cell and tissue sources. Such data are central to our understanding of functional glycomics, and this knowledge can also be used for pathway construction and other applications in the field of Systems Glycobiology. Glycans released from cell/tissue samples are often studied in their free-form. They can also be functionalized with aglycones like 2-aminobenzamide (2AB) and procainamide to enhance separation and improve ionization during liquid chromatography/mass spectrometry. Additionally, these released glycans may be permethylated in order to improve glycan quantitation. In such work, besides studying the glycans in a single sample, there is also interest in comparing multiple samples in order to determine underlying similarities and differences, for example in terms of specific epitopes that are changed when cells of the same origin differentiate along different pathways. The current chapter describes the development and usage of cGlyco (“comparative Glycomics”), an open-source program that can be used to compare data from multiple mass spectrometry runs. As an example, we apply cGlyco to compare the glycan profile of multiple MALDI-TOF glycomics profiling data collected by core-C of the Consortium for Functional Glycomics (CFG).