The aim of our study was to optimise electric pulse parameters for electrochemotherapy by sampling the space of pulse parameter variables using systematic in vitro experiments. For this purpose we defined parameters that describe the effectiveness of different sets of electric pulse parameters in vitro and combined them into an objective function that characterises requirements for successful electrochemotherapy. The objective function values were calculated for all the sets of electric pulse parameters included in in vitro experiments. Similar values were grouped together by hierarchical clustering. The 'electrochemotherapeutic' effectiveness of two sets of pulse parameters (8 pulses, 100 micros, 1 Hz and 1 pulse, 1000 micros, 1 Hz), which belong to the most efficient cluster, and one set of pulse parameters (16 pulses, 20 ms, 1 Hz), which belongs to the least efficient cluster, was tested in vivo on a murine tumor model. The sets of pulse parameters from the most efficient cluster had comparable effects in vivo, while the electrochemotherapy with the set of pulse parameters from the least efficient cluster was less effective. Our results demonstrated that electric pulse parameters for effective in vivo application can be determined from in vitro experiments considering application specifications.