The aim of this study was to report on a new technique for examining aqueous humor. Aqueous humor samples obtained from 198 patients (cataract: n=66 eyes; age-related macular degeneration [AMD]: n=66; diabetic macular edema: n=66) were in vitro examined by mid infrared spectroscopy. After training and validating the technique, 66 samples were taken for the final independent prediction. In computerized analysis of the spectrometric data, 22 (100%) cataract eyes were diagnosed as the cataract group, 17 (77%) diabetic macular edema eyes were diagnosed as the diabetic macular edema group, and 14 (64%) eyes with AMD were diagnosed as the AMD group. Mid infrared spectrometry could statistically significantly (P=0.004) differentiate between the 3 study groups. Mid infrared spectroscopic examination of aqueous humor may be able to differentiate eyes with cataract, AMD, or diabetic macular edema.