The modulation transfer function (MTF) describes how an imaging system modifies the spatial frequency content of a scene. Many performance metrics and specification requirements are strongly dependent on the MTF as it provides information on the limiting resolution of the imaging system. In this correspondence we will identify potential issues that can contribute uncertainty or bias into the MTF measurement, and suggest best practices to avoid such issues. Beginning with a full 2D derivation of the tilted edge measurement technique, we identify potential areas where differences between laboratories can occur due to the setup, imaging system, measurement procedure, or the measurement processing. We show specific examples of how the system’s nonuniformity (including defective pixels) can affect the observed MTF. Additionally, we show examples of target to target variation and the effects of dynamic range. A summary table is provided on best practices to reduce the impact of the identified potential areas of difference. In support of the reproducible research effort, the Matlab functions associated with this work can be found on the Mathworks file exchange [1].