Two acyl hydrazone derivatives, AHI and AHN, made from ibuprofen and naproxen-derived hydrazides, were prepared and studied of binding properties with serine protease trypsin by UV-vis absorption and fluorescence quenching at pH 7.4. The results suggest that both hydrazones can interact strongly with trypsin and there are the formation of trypsin-hydrazone complexes. The Stern-Volmer constants, binding constants, binding sites and the corresponding thermodynamic parameters Delta H-o, Delta S-o and Delta G(o) were calculated at different temperatures. The effect of common metal ions on the constants was also discussed. The binding modes can be explained on the basis of hydrogen bonds and van der Waals forces. The binding distance (r) similar to 3 nm between the donor (trypsin) and acceptors (AHI and AHN) was obtained according to Forster's non-radiative energy transfer theory. Moreover, LOD and LOQ of hydrazones were calculated in the presence of trypsin.