The trans-Golgi protease furin is a protein convertase playing crucial roles in homeostasis, and in diseases ranging from anthrax and Ebola fever to Alzheimer’s disease and cancer 1. Increase of furin in tumors correlates with the increase of membrane type 1-matrix metalloproteinase (MT1-MMP), which activates extracellular pro-MMP2 to induce rapid tumor growth and metastasis 2. Therefore, noninvasive imaging of furin activity offers a valuable tool to probe tumor growth and migration in real time and directly assess the anti-cancer efficacy of drugs in vivo 3. It has been reported that the majority of human breast cancer cells overexpress furin 4.Traditional immunofluorescence staining of MDA-MB-468 cells indicates that furin is predominantly located in the trans-Golgi networks of this type of breast cancer cells 5. While there are very few methods that have been reported to image furin activity directly, Rao and coworkers developed two methods of intracellular condensation and intramolecular macrocyclization for imaging furin activity in living cells using fluorescence probes 6-7. Two-photon laser microscopy (TPLM) is a fluorescence imaging technique that allows imaging of living tissues up to a very high depth. It uses red-shifted excitation light to excite fluorescent dyes. For each excitation, two photons of the infrared light are absorbed simultaneously. TPLM can be a superior technique due to its deep tissue penetration, efficient light detection and reduced phototoxicity 8. Furin preferentially cleaves Arg-X-Lys/Arg-Arg↓X motifs, where Arg is arginine, Lys is lysine, X can be any amino acid residue and ↓indicates the cleavage site 9. Combining these two advantages above, recently we developed Acetyl-Arg-Val-Arg-Arg-Cys(StBu)-Lys(Eu-DOTA)-CBT (2) for imaging furin-controlled condensation in MDA-MB-468 cells (Fig. 1) 10. Its scrambled control, 2-Scr, was studied in parallel. In brief, 2 contains a RVRR peptide sequence for furin cleavage and cell membrane translocation, disulfided Cys for supplying the 1,2-aminothiol group for condensation with the cyano group on the benzothiazole motif, Lys conjugated with Eu-DOTA for TPLM. With the probes designed, we successfully imaged the furin-controlled intracellular condensation of 2, as well as the location and activity of furin (Fig. 2). We describe herein a detailed protocol of cell preparation and TPLM imaging with 2.