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Human serum gangliosides were analyzed by nano-HPLC/MS to characterize their glycoforms and ceramide profiles within a single experiment. This unbiased glycosphingolipidomics approach was successfully applied to a set of clinical samples of gastric and pancreatic cancer patients and related control cohorts. Evaluation of acquired data revealed no clear differences with respect to ganglioside glycoforms among the patient groups investigated. However, significant alterations were observed within the ceramide profiles of different ganglioside structures, particularly pronounced for gangliosides GM2 and GD1a. Based on the increase of palmitic acid-comprising structures and the concurrent decrease of stearic acid-comprising ganglioside signals observed for both glycoforms, the ceramide ratio of these signals was applied for discrimination of cancer patients from control sample cohorts. Based on the performance evaluated in statistical analyses, the GM2-related ceramide ratio is proposed as a potential biomarker in particular for pancreatic cancer. Although further validation is required to determine its capability for cancer detection in mass screenings and its applicability for clinical routine, the data presented here support a promising perspective for applications.