A new fluorogenic trimethyl lock quinone (TLQ) derivative, designated as the quencher-TLQ-fluorophore-type probe (Q-TLQ-F), was developed for selective detection of nicotinamide adenine dinucleotide phosphate (NADPH). By taking advantage of the well-known facile intramolecular ring cyclization reaction (δ-lactonization) of TLQ that can release a reporter molecule upon reduction, Q-TLQ-F was successfully applied for the detection of physiological NADPH generated by glucose-6-phosphate dehydrogenase in human breast cancer MDA-MB-231 cells.