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Background: Sensitive and rapid diagnosis of the early stages of glaucoma from tear fluid is a great challenge for researchers. Methods: Tear fluid was analyzed using three-dimensional synchronous fluorescence spectroscopy (3D-SFS). Our previously published results briefly describe the main methods which applied the second derivative to a selected synchronous spectrum Δλ = 110 nm in distinguishing between healthy subjects (CTRL) and patients with glaucoma (POAG). Results: In this paper, a novel strategy was used to evaluate three-dimensional spectra from the tear fluid database of our patients. A series of synchronous excitation spectra were processed as a front view and presented as a single curve showcasing the overall fluorescence profile of the tear fluid. The second derivative spectrum provides two parameters that can enhance the distinction between CTRL and POAG tear fluid. Conclusions: Combining different types of 3D-SFS data can offer interesting and useful diagnostic tools and it can be used as input for machine learning and process automation.