Optical trapping is an experimental technique used to apply piconewton scale forces onto model membrane systems. This allows us to measure changes in key physical properties as membranes undergo dynamic processes such as shape changes. To obtain accurate measurements of force, the “stiffness” of the trap must be determined through a calibration step. The equipartition calibration method calculates the stiffness by algorithmically tracking the position of a trapped bead over time. Camera readout noise creates inaccuracies in tracking which are propagated to the calibration step. We describe a mathematical model to correct for such inaccuracies and obtain a better calibration estimate. We use simulations based on empirical image data to determine the parameters in this model, and we demonstrate that our model indeed leads to improved calibration. With this new equipartition calibration method, we will be able to adapt our optical trapping setup to perform experiments investigating how the insertion and removal of lipids and proteins in model cell membranes affects membrane properties. These studies ultimately provide insight into the interplay between the multitude of processes occurring at the cell membrane.