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In general, 4 amino-5-methylamino-2′,7′-difluorescein diacetate (DAF-FM-DA) dye is used to detect nitric oxide in biological systems through cell imaging. In this study, we have used 96 well plate format to quantify nitric oxide using DAF-FM-DA through a multimode reader (or independently using fluorospectrometer) and could be visualized in a fluorescence microscope. Similar study otherwise will require a high-end instrument. The method has been validated to screen NOS inhibitors in the HEK 293T cell lines over-expressing the NOS isoforms. We observed that the method is very simple to use, adaptive, sensitive and most importantly it saves time. Ethanol (70% [v/v] in distilled water), Nω-Nitro- l -arginine (l -NAME), 7-Nitro-Indazole (7-NI) (Sigma, St. Louis, MO), HEK 293T cell lines (National Centre for Cell Science (NCCS), Pune, India), DMEM (Himedia laboratories Pvt), Fetal Bovine Serum (FBS) (Invitrogen, Carlsbad, CA), 100 U/mL penicillin, and 0.1 mg/mL streptomycin in a 5% CO2 atmosphere. Hank's Balanced Salt Solution (HBSS) without Phenol Red of pH 7.4 was prepared with the following composition: NaCl, 8.0g, KCl, 0.4g, CaCl 2 , 0.14g, MgSO 4 ⋅7H 2 O, 0.1g, MgCl 2 ·6H 2 O, 0.1g, Na 2 HPO 4 ·2H 2 O, 0.06g, KH 2 PO 4 , 0.06g, glucose, 1.0g, NaHCO 3 , 0.35g, H 2 O, to 1000 ml, Sterilized and refrigerated, Calcium Ionophore A23187 (Sigma Aldrich 52665-69-7) DAF-FM Di Acetate (Molecular Probes Life Technologies), and DAF-FM Di Aceatate was prepared as a stock solution (5 mM) in DMSO, divided into aliquots and stored at −20 °C, followed by dilution to the required concentration in HBSS buffer before use. Neubauer chamber, Microtube centrifuges (1.5 mL), Micropipettors,10,100, and 1000 mL with corresponding tips, multimode reader (Tecan, Synergy-HT), inverted fluorescence microscope (Nikon, eclipse Ti-S), black flat bottom Microplates (96-well) (Corning 3603). Image 1 • A simple, sensitive and adaptive cell-based protocol that requires a microplate reader screen the potential NOS inhibitors. • Enhanced quantum fluorescence of DAF-FM-DA with intracellular NO improves its sensitivity and enzyme activity. • This cell-based assay provides better insight into the bioavailability and inhibitory activity of the potential compounds. [ABSTRACT FROM AUTHOR]