An autofocus system adaptable for use in microscopes or other optical systems incorporates an auxiliary photodetector that measures light only from a reference light source. An outgoing reference beam, incident upon a target to be inspected, is reflected to become an incoming reference beam incident on a photodetector. The photodetector generates a focus error signal proportional to the displacement of the target from the ideal focal point. The auxiliary photodetector generates a correction signal proportional to the output of the reference light source. The correction signal is used to compensate the focus error signal for stray light generated by the reference light source. The compensated focus error signal may be used to drive a servomechanism to displace either the target or the microscope objective lens to bring the target into focus. The correction signal may also be used to compensate the reference light source gain to control the reference light source output.