Objective: The aim of this study was to report on a new technique for examining aqueous humor. Participants: 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. Results: 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. Conclusions: Mid infrared spectroscopic examination of aqueous humor may be able to differentiate eyes with cataract, AMD, or diabetic macular edema.