Choline kinase alpha (ChoKa) deregulation is associated with a more aggressive phenotype and greater malignancy in many human cancers. Inhibitors of ChoKa induce apoptosis in tumorigenic cells, but validation of their specificity is difficult in vivo. Alterations in the profile of choline metabolites are detectable by magnetic resonance spectroscopy (MRS), but because of competing catabolic contributions from the phospholipases, the relative role of ChoKa is not absolutely discernable in a clinical setting. The goal of this work was to develop ChoKa-specific imaging probes to assist in the development of ChoKa as a diagnostic biomarker and therapeutic target. A series of compounds were synthesized for this purpose, and JAS239 was identified as the most promising choline-mimetic with inherent near infrared fluorescence. Attenuation of choline phosphorylation by JAS239 in human breast cancer cells was observed using 14C-choline radiotracing and high-resolution 1H MRS. Microscopy was used to explore the interaction of JAS239 with the ChoKa protein. These in vitro studies, using the established MN58b as a positive control, indicated that JAS239 functions as a competitive inhibitor of ChoKa. Athymic nude mice inoculated with human breast cancer xenografts were injected i.v. with trace doses of JAS239 for imaging studies. In vivo optical imaging of JAS239 accumulation delineated breast tumor margins, and the signal intensity was capable of distinguishing both genetic overexpression and pharmacologic inhibition of ChoKa in breast xenografts. At therapeutic doses, JAS239 and MN58b reduced murine xenograft growth rates, and JAS239 was more effective than MN58b at reducing tumor total choline levels. Histological assessment found both JAS239 and MN58b reduced tumor cell density, decreased proliferation, and elicited an apoptotic response. In a parallel study, ChoKa inhibition was shown for the first time to be an effective therapeutic strategy in glioma tumors, however, JAS239 was not found to cross the blood-brain barrier. A library of derivatives were synthesized and these are being investigated to improve the potency, biodistribution, and tumor specificity. These results represent a new paradigm of multifunctional small molecules that can be used for ChoKa measurement, as companion diagnostics to validate new ChoKa inhibitors, and as therapeutically-effective inhibitors of choline metabolism.