The intrinsic characteristics of many tropical fruits (thin peel, high content of water and low molecular weight compounds) cause high post-harvest losses and impede their commercialization as fresh fruits. Beyond the interest for bioactive compounds present in the fruits, the full fruit is a good source of carbohydrates and fibers. Processing of tropical fruits into products with higher stability and shelf life has the potential to significantly reduce the waste and increase their utilization. Information about the composition of the fruits is crucial for defining processing conditions and identifying opportunities for product development. The analytical methods generally used to quantify sugars and organic acids are costly and time-consuming. Simultaneous analysis by liquid chromatography-mass spectrometry (LC-MS/MS) is a very sensitive and reproducible option, giving accurate simultaneous multi-analyte quantification in complex systems. We established a LC-MS/MS multiple reaction monitoring (MRM) method using reverse column for detecting and quantifying sugars (glucose, fructose and sucrose) and organic acids (citric, malic and tartaric acid) in only 4 minutes, in selected tropical fruits: araçá (Psidium cattleianum), ceriguela (Spondias purpurea), manga (Mangifera índica), goiaba (Psidium guajava) and pitanga (Eugenia uniflora L.). We have found that the sucrose content varied from non-detected for pitanga up to 64.54 mg/g for mango, indicating a large variation in sugar composition between the fruits. All fruits had similar concentration of glucose and fructose (~27 mg/g), with the exception of pitanga. Some fruits stood out by their high content of organic acids: araçá and pitanga were rich in citric acid (~6.6 mg/g) and pitanga in malic acid (21.6 mg/g). The newly generated data on the composition of tropical fruits allow to establish a correlation between the carbohydrate content and the physical properties of the fruit pulps, helping product and process development.