The paradigm in bio-AFM is to measure a few dozen of cells and to draw fundamental biophysical conclusions. To apply these results in biology or medicine, it is essential to increase the statistics by measuring of significant number of cells and to automate the measurements. In this context, it is necessary to optimize the measurements, to reduce the results dispersity and to increase the experiment speed. Concerning the dispersity, we hypothesize that cells organized on patterns would have reduced Young’s modulus dispersion. We had the same hypothesis in regard to the indenter shape (cone vs. colloidal probe). Figure 1 shows that randomly shaped cells (A) present Young's moduli more dispersed than cells that have been forced into a square shape (B). The use of patterns therefore makes it possible to increase the homogeneity of the obtained results. We tested the effect of an indenter which probes a larger surface with a 5µm-diameter colloidal probe. The Young's modulus is smaller with the colloidal probe than with the pyramidal probe, but the dispersion is also smaller. There is therefore an advantage to use a colloidal probe in the automation process. To increase the measurement speed, we worked on two parameters, the tip velocity and the number of indentations per cell. We therefore evaluated the impact of the indentation velocity on the resulting mechanical properties. Young's modulus is higher with increasing velocity. But this trend is comparable for cells constrained by square patterns of fibronectin and those not constrained. A high indentation velocity can therefore be applied during automation. Finally, we compared the influence of the number of indentations (1,024; 256; 64 and 16) per cell . There is no significant difference in Young's modulus value between each condition. Automation can therefore be done with a reduced number of indentations per cell. To conclude, this optimization work opens the door to the implementation of automatic measurements on a large number of cells, which will allow us to probe a large number of cells in a controlled time. This will open the bio-AFM to statistical analyses on populations of cells of interest.