Various conventional methods have been reported for in vitro detection of NADH, such as, liquid chromatography-mass spectrometry, capillary electrophoresis, electrochemical analysis and colorimetric method. In recent time, fluorescence imaging is considered as an efficient method in biomedical research due to its high specificity, short response time, ease of handling, and high sensitivity. Considering the advantages of fluorescence imaging, different fluorescent probes have been widely designed and used for detecting and imaging intracellular NADH levels. Furthermore, the intrinsic fluorescence of NADH has been used for imaging and quantification of various biomolecules. NADH absorbs light at 340 nm and emits at 460 nm, but due to its short excitation as well as emission wavelength, it is easily interfered with other biomolecules in the cell such as amino acids. Thus, it is required to employ fluorescent probes with fluorescence emission at higher wavelengths to avoid fluorescence interference from NADH fluorophore.