Conventional method of calibrating optical trap stiffness is applicable for microspheres whose diameters range from hundreds of nanometer to several micrometers, but only have a slight advantage for those microspheres with diameters lager than five micrometers. To compensate this, we experimentally develop a time of flight method for measuring optical trap stiffness with larger microspheres. By comparing the optical trap stiffness of microspheres with different sizes and different materials at different laser powers, the time of flight method is confirmed to be more accurate and practical for microspheres larger than 5 μm; the result is of the same order of magnitude as the results of Brownian noise based analysis of 5 μm polystyrene bead. The results are higher than theoretical values due to the limited bandwidth of the camera. In comparison, the time of flight method is superior to other methods and does make sense in the fast calibration of optical trap stiffness on cell level. This method can be applied to optical traps with special field distributions. In the measurement of mechanical properties of cells, it can avoid using microspheres as force probe, thus providing a novel approach to the study of sophisticated single molecule process on the membrane of cells.