In this paper, we report a calibration of acoustic trapping force of single-beam acoustic tweezer (SBAT) at ultrahigh frequency using micropipette aspiration. The acoustic trapping forces ( Ftrapping) and the trap stiffness on a 5- [Formula: see text] polystyrene microbead for a 110-MHz SBAT were measured against the known force generated from a micropipette. The trap stiffness ( k ), which represents Ftrapping corresponding to a displacement ( x ) of a microbead from the trap center, was measured and the results showed that a higher duty factor and excitation voltage lead to a stronger trapping force and trap stiffness for a given displacement. Since a precisely calibrated force generated from a micropipette is directly applied to the calculation of acoustic trapping force, the approach should be more flexible than those previously reported. In addition, with this method, precisely controlling the tip size of a micropipette within a few micrometers allows the possibility of calibrating the trapping force on an object of the size of a single cell. It not only helps better evaluate the trapping performance of SBAT as a tool of cell manipulation, but also helps develop SBAT as a useful tool for assessing cellular interactions.