This work presents the immobilization of trypsin on the synthesized pillar[5]arene derivative (P5) containing ten iodo-terminals. The characterization has been carried out by Thermogravimetric Analysis (TGA), Fourier Transform Infrared (FTIR) and Raman spectra, Scanning Electron Microscopy (SEM) and Zeta Potential. Furthermore, Cytochrome c (Cyt c) was chosen as a model protein for evaluation of the performance of the pillar[5]arene-immobilized trypsin (P5-T), and its microwave-assisted digestion conditions were investigated by using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for 15 second. The peptide numbers of 10 and sequence coverage of 93% for 15 second microwave-assisted digestion were obtained for P5-T. The results can be further applied in future proteomics studies due to high efficiency, good reproducibility and stability of the pillar[5]arene-immobilized trypsin.