Molecular optical imaging in vivo has potential for functional assessment of tumor tissue at the cellular and subcellular level. Advances in biological understanding of genomics and proteomics have increased the knowledge of possible cellular targets, allowing for functional detection, diagnosis, characterization and treatment monitoring of disease in situ. Yet current cancer treatment monitoring largely relies on structural imaging to quantify tumor size change, which can occur well after many identified molecular events have transpired. In this work, three model glioma cell lines were implanted orthotopically, including a green fluorescent protein (GFP) expressing rat gliosarcoma (9L-GFP), a human glioma (U251) and the GFP variant of this (U251-GFP). Tumor morphology was assessed through magnetic resonance imaging (MRI) and ex vivo pathology, which confirmed distinct phenotypic growth patterns of the three tumor models. The tumors were non-invasively monitored using an optical transmission spectroscopy system targeted at imaging tumor cell metabolism and EGFR activity with near-infrared light. Monitoring of tumor cell metabolism was accomplished through detection of Protoporphyrin IX (PpIX) which is formed via the Heme synthesis pathway from the administered prodrug, Aminolevulinic Acid (ALA), and assesses mitochondrial activity. The molecular expression of epidermal growth factor receptor (EGFR) was quantified using an infrared dye (IRDye 800CW) conjugated to epidermal growth factor (EGF). Monitoring tumor cell metabolism and molecular expression of EGFR via transmission spectroscopy provided early functional information of in vivo tumor tissues which could be correlated to later occurring structural changes seen by MRI. Animals were treated with Erbitux (Cetuximab), a monoclonal antibody therapy which inhibits EGFR, and monitored noninvasively for response to therapy via fluorescence signature changes, which corresponded to changes in uptake of EGF conjugated to IRDye 800CW. Fluorescence signatures attained for populations of mice for both PpIX and EGF conjugated IRDye 800CW were variable highlighting the heterogeneity of cancer as a disease. Functional monitoring of glioma tissue was achieved with noninvasive nearinfrared transmission spectroscopy and will increase the ability of molecular imaging strategies to interpret cancer as an individual disease. Throughout this work, there were several technological strategies for optimal measurement of the signal developed which included transmission based spectroscopic imaging, normalization of the transmitted fluorescence signal to the transmitted excitation signal and spectral fitting of the fluorescence signal to deconvolve the fluorescent signal from any non-specific background signal. In this work these strategies were optimized to attain the most possible signal and incorporated into a prototype monitor that would allow widespread use in cancer screening and monitoring.