This study deals with quantifying sugar and acids levels important for the perceived taste of tomatoes (Solanum lycopersicum). Sugar and acids levels were measured repeatedly on the same tomato using tissue samples obtained with a biopsy needle in combination with HPLC protocols. Biopsies of pericarp and locular gel tissue from tomatoes differing in position in the truss, from mature green to ripe red, were taken from a beef- (‘Licorossa’), a cocktail- (‘Lucino’) and a cherry type (‘Petit Sweet’) cultivar. Tomatoes were stored up to three weeks at three temperatures (12, 19 and 24.5 °C) and biopsy samples were taken every few days. A model regarding the most important processes that interconvert sugars and acids (glycolysis, TCA cycle and gluconeogenesis (GNG)) is proposed. Results of the model calibration showed more breakdown of hexoses in red tomatoes and more conversion of malate into hexoses in green tomatoes. More hexose turnover was found in locular gel than in pericarp tissue. GNG was more important in the cherry type cultivar due to faster hexose and malate breakdown. In the round type cultivar malate levels were higher due to faster citrate breakdown and slower malate breakdown. Starch and sucrose levels did not significantly affect postharvest sugar and acid development. Molecular markers that quantify the kinetic parameters of the model might be important to develop genotypes with better taste performance.