Optimization of delactosed whey permeate (DWP) treatment for fresh-cut tomato was accomplished by evaluating different quality, nutritional and microbial markers. Response surface methodology was applied to obtain polynomial model equations. DWP concentration (0-5%) and storage (0-10 days) were used as independent factors in order to optimize the process. The analyses showed that increases in DWP concentration extended the quality of the fresh-cut tomato significantly (p<0.05) by maintaining texture and antioxidant activity (FRAP) and controlling the spoilage during the storage. The total aerobic counts and yeast and molds were reduced by ∼1.5 log cfu/g and ∼1.0 log cfu/g respectively after 10 days of storage treated with 3% DWP. Ascorbic acid and lycopene were retained best within the range of 3 to 5% of DWP treatment. However, concentrations>3% were scored unacceptable by the sensory panel due to perceived off-odors. Predicted models were highly significant (p<0.05) for all the markers studied in fresh-cut tomato with high regression coefficients (R2) ranging from 0.79 to 0.99. The study recommends the use of DWP at a concentration of 3% to extend the shelf life of fresh-cut tomato by preserving its quality and antioxidant properties during storage.