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Organic acids are important active small molecules present in venoms and toxins, which have not been fully explored yet. The aim of the study was the determination of organic acids in honeybee venom (HBV) samples by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Two protocols for sample preparation were employed. A solid-phase extraction was used for the determination of malonic acid, fumaric acid, glutaric acid, and kynurenic acid. A dilute-and-shoot method was optimal for: citric acid, malic acid, and succinic acid. Chromatographic separation was performed using a Synergi Hydro-RP column. Detection was performed on a triple-quadrupole mass spectrometer operating in multiple reaction monitoring mode. Among the analytes, glutaric acid and kynurenic acid were present in HBV samples in the lowest concentrations, whereas citric acid was the most abundant acid in each sample, and accounted for an average of 86 mg/g (8.6%) of the venom dry weight. Organic acids were discussed in terms of function. This is the first study in the available literature that provides specific data on the content of organic acids in HBV using a validated quantitative method.