The interaction between indomethacin and human serum albumin (HSA) was investigated by fluorescence quenching technique and UV–vis absorption spectroscopy. The results of fluorescence titration revealed that indomethacin, strongly quench the intrinsic fluorescence of HSA by static quenching and nonradiative energy transfer. The binding site number n and the apparent binding constant KA, were calculated using linear and nonlinear fit to the experimental data. The distance r between donor (HSA) and acceptor (indomethacin) was obtained according to fluorescence resonance energy transfer (FRET). The study suggests that the donor and the acceptor are bound at different locations but within the quenching distance.