Binding force between biomolecules has a crucial role in most biological processes. Receptor-ligand interactions transmit physical forces and signals simultaneously. Previously, we employed a robotic micropipette both in live cell and microbead adhesion studies to explore the adhesion force of biomolecules such as cell surface receptors including specific integrins on immune cells. Here we apply standard computational fluid dynamics simulations to reveal the detailed physical background of the flow generated by the micropipette when probing microbead adhesion on functionalized surfaces. Measuring the aspiration pressure needed to pick up the biotinylated 10 μm beads on avidin coated surfaces and converting it to a hydrodynamic lifting force on the basis of simulations, we found an unbinding force of 12 ± 2 nN, when targeting the beads manually; robotic targeting resulted in 9 ± 4 nN (mean ± SD). We measured and simulated the effect of the targeting offset, when the microbead was out of the axis (off-axis)of the micropipette. According to the simulations, the higher offset resulted in a higher lifting force acting on the bead. Considering this effect, we could readily correct the impact of the targeting offset to renormalize the experimental data. Horizontal force and torque also appeared in simulations in case of a targeting offset. Surprisingly, simulations show that the lifting force acting on the bead reaches a maximum at a flow rate of ~ 5 μl/s if the targeting offset is not very high (<5 μm). Further increasing the flow rate decreases the lifting force. We attribute this effect to the spherical geometry of the bead. We predict that higher flow rates cannot increase the hydrodynamic lifting force acting on the precisely targeted microbead, setting a fundamental force limit (16 nN in our setup) for manipulating microbeads with a micropipette perpendicular to the supporting surface. In order to extend the force range, we propose the offset targeting of microbeads.