This paper describes an error separation method for a precision measurement of the run-out of a microdrill bit by using a measurement system consisting of a concentricity gauge and a laser scan micrometer (LSM). The proposed error separation method can achieve a sub-micrometric measurement accuracy of the run-out of the microdrill bit without the requirement of ultra-precision rotary drive devices. In the measurement, the spindle error motion of the concentricity gauge is firstly measured by using the LSM and a small-diameter artifact, instead of the conventionally used displacement probes and large-diameter artifact, in order to determine the fine position of the concentricity gauge when the spindle error motion is at its minimum. The microdrill bit is rotated at the fine position for the measurement of the run-out, so that the influence of the spindle error motion can thus be reduced, which could not be previously realized by commercial measurement systems. Experiments were carried out to verify the feasibility of the proposed error separation method for the measurement of the run-out of the microdrill bit. The measurement results and the measurement uncertainty confirmed that the proposed method is reliable for the run-out measurement with sub-micrometric accuracy.