Post translational modification of proteins takes place via glycosylation, and changes in glycan structure, which can be associated with biological function, are seen in malignancies. Efficient assessment of glycans, particularly in clinical settings, can be hampered by specimen size, and lengthy sample preparation. We have developed an effective procedure for N-glycan analyses using chloroform-methanol (CM) extraction of specimens in the absence or presence of water (CMW), prior to enzymatic cleavage with PNGase F. This procedure was used to determine glycan profiles in cancer cell lines, and biopsies from lung cancer. Subsequently, to demonstrate the broad applicability of the method, cancer cell lines originating from other types of tumors were also studied, including non-Hodgkin B-lymphoma, choriocarcinoma and histiocytoma. The method was successfully applied to investigation of N-glycans from small numbers of in vitro cultured cells (≤1×105) and to tumor tissues, including patient biopsies of small size. MALDI-MS analysis confirmed the efficient release of all N-glycan types, including complex forms with poly-N-acetyllactosamine chains. Importantly, in patient biopsy specimens and others, the non-aqueous CM extraction yielded high-mannose glycans with one GlcNAc moiety, suggesting preservation of an Endo-H like enzyme activity. This method enables practical application of glycan profiling to small clinical specimens, as well as detection of Endo H-like enzymatic activities in cancer cells, which is a previously unrecognized phenomenon.