A two-dimensional or transverse acoustic trapping and its capability to noninvasively manipulate micrometersized particles with focused sound beams were experimentally demonstrated in our previous work. To apply this technique, as in optical tweezers, for studying mechanical properties of and interactions among biological particles such as cells, the trapping forces must be calibrated against known forces, i.e., viscous drag forces exerted by fluid flows. The trapping forces and the trap stiffness were measured under various conditions and the results were reported in this paper. In the current experimental arrangement, because the trapped particles were positioned against an acoustically transparent mylar membrane, the ultrasound beam intensity distribution near the membrane must be carefully considered. The total intensity field (the sum of incident and scattering intensity fields) around the droplet was thus computed by finite element analysis (FEA) with the membrane included, and it was then used in the ray acoustics model to calculate the trapping forces. The membrane effect on trapping forces was discussed by comparing effective beam widths with and without the membrane. The FEA results found that the broader beam width, caused by the scattered beams from the neighboring membrane and the droplet, resulted in the lower intensity, or smaller force, on the droplet. The experimental results showed that the measured forces were as high as 64 nN. The trap stiffness, approximated as a linear spring, was estimated by linear regressions and found to be 1.3 to 4.4 nN/μm, which was on a larger scale than that of optical trapping estimated for red blood cells, a few tenths of piconewtons/nanometer. The experimental and theoretical results were in good agreement.