Recently, we pioneered the application of Cherenkov radiation (CR) of radionuclides for the in situ excitation of discrete Eu(III) and Tb(III) complexes. CR is produced by isotopes decaying under emission of charged particles in dielectric media and exhibits a maximum intensity below 400 nm. We have demonstrated that luminescent lanthanide antenna complexes are ideal acceptors for Cherenkov radiation-mediated energy transfer (CRET). Here, we develop and assess peptide-functionalized Tb(III) and Eu(III) complexes in conjunction with CRET excitation by the positron emissive radioisotope 18F for simultaneous, multiplexed imaging and in vivo optical imaging. This work demonstrates, for the first time, that the detection of the luminescence emission of a discrete Eu(III) complex in vivo is feasible. Our results open possibilities for discrete luminescent lanthanide complexes to be used as diagnostic, optical tools for the intrasurgical guidance of tumor resection.