PURPOSE: To introduce and validate quantitative oblique back-illumination microscopy (qOBM) as a label-free, high-contrast imaging technique for visualizing conjunctival goblet cells (GCs) and assessing their functional changes. METHODS: qOBM was developed in conjunction with moxifloxacin-based fluorescence microscopy (MBFM), which was used for validating GC imaging. Initial validation was conducted with polystyrene beads, followed by testing on normal mouse conjunctiva under both ex-vivo and in-vivo conditions. Longitudinal qOBM imaging was performed on ex-vivo mouse conjunctiva exposed to hyperosmotic stress (induced by 1000 mOsm/kg NaCl solution) and normal saline (300 mOsm/kg balanced salt solution, BSS). Imaging was conducted at baseline and at 15- and 30-min instillation. Results were compared to those of MBFM and periodic acid-Schiff (PAS) staining. A similar longitudinal study was performed in-vivo, and the outcomes were analyzed. RESULTS: qOBM accurately imaged polystyrene beads, with measured phase delays matching theoretical predictions. In normal mouse conjunctiva, qOBM visualized GCs in high contrast, confirmed by MBFM, and the average phase delay was 0.59 ± 0.25 radians. Under hyperosmotic stress, qOBM detected a significant reduction in GC phase delays, decreasing to levels of the surrounding tissue (-0.07 ± 0.14 radians). In normal conditions, no notable changes were observed in GCs. In-vivo imaging results were consistent with ex-vivo findings. Statistical analysis further characterized these changes. The results were consistent with MBFM and PAS staining. CONCLUSIONS: qOBM is a high-contrast, label-free imaging modality that enables the functional assessment of GCs. This technique holds significant potential for advancing research and clinical diagnostics related to ocular surface diseases.