This thesis presents an optimized triple modality reporter combining genes for a far-red fluorescent protein (E2-Crimson), enhanced firefly luciferase enzyme (Luc2), and truncated wild type herpes simplex virus I thymidine kinase (wttk). This schematic allows for sensitive, long-term tracking of tumor growth in vivo by fluorescence, bioluminescence, and positron emission tomography. This triple reporter improves on previous designs in three ways. 1) The shorter wavelength green fluorescent protein (GFP) or monomeric red fluorescent protein (mRFP1) has been replaced with E2-Crimson to increase penetration of fluorescence signal through mammalian tissues. 2) Firefly luciferase (fLuc), Renilla luciferase (hrl), or mutant thermally stable firefly luciferase (mtfl) has been replaced by Luc2, a codon optimized firefly luciferase for increased expression in mammalian cells and detection of single cells by bioluminescence. 3) Self-cleaving viral 2A sequences separate each component, ensuring equal stoichiometry of the reporter genes without requiring protein fusion or the use of internal ribosomal entry site (IRES) sequences. Cleavage between each protein gene product allows for proper protein folding, trafficking, and full activity of each modality. This optimized triple reporter construct was cloned into a second-generation lentiviral vector, and a lentivirus was produced. Two human cancer cell lines were virally transduced with the triple reporter construct, and expression of all three modalities was confirmed in both cell lines in vitro and in vivo. Finally, the therapeutic responses of the MDA231 human breast cancer cell line to the chemotherapeutic agents monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF) were successfully quantified in vivo by optical imaging of the optimized triple modality reporter. This is the first reported use of both fluorescence and bioluminescence signals from a multi-reporter construct to measure drug efficacy in vivo.