Adenosine triphosphate (ATP) plays a significant role in storing the energy required for biological processes in living cells – synthesis, transport, mobility of molecules and more. The energy is stored in the bonds between phosphate groups, and the structure of the ATP molecule consists of a chain of three phosphate groups along with D-ribose and adenine. The necessity for ATP has increased with the application of extracellular enzymatic processes for different industrial purposes, such as production of drugs, vitamins, food, textiles and others. Due to its high cost and frequent usage, increasing attention has been focused on developing novel pathways for its regeneration. This paper describes the process of ATP synthesis from adenosine and adenosine monophosphate (AMP) through a linear cascade reaction using the enzymes ADK, PPK2-I, PPK2-II. The experiment was conducted using mathematical modeling and genetic algorithm with computer programs SCIENTIST and GALOP (Genetic Algorithm for the Optimization of Processes). In SCIENTIST, a simulation of ATP synthesis was conducted, while GALOP was used to optimize the initial conditions of process – concentration of adenosine, AMP, magnesium ions and reaction duration. The concentrations of enzymes and polyphosphate were kept constant. The aim of this study was to find the optimal process conditions to achieve the maximum concentration of ATP. The results obtained showed that, under optimal conditions, it is possible to achieve an ATP concentration of 20,7 mM.