Artifacts related to the wave-spreading phenomenon of fluorescent structures within the eye interfere with interpretation of vitreous fluorophotometric data. A standardized protocol which minimizes such artifacts is desirable in order to reveal actual changes in vitreous fluorescence. We have devised a protocol which reduces artifacts of crystalline lens autofluorescence and“tailing”from both lens and retina by the subtraction of preinjection data from measurements taken one hour after intravenous injection of fluorescein. A method of calculation is presented, performed automatically when programmed into a computerized fluorophotometer. Distinctions among healthy subjects and different kinds of ocular pathology are sharper when artifacts are reduced and the data are normalized to plasma fluorescein concentration. Examples of data from subjects with diabetes and no retinopathy, hypertension, cystoid macular edema, panuveitis and pars planitis are presented.