The comprehensive work of both clinical and basic science colleagues has demonstrated a clear proof of concept for “in vitro discovered- in vivo validated” biomarkers in translational metabolic profiling research using magnetic resonance techniques. Major tissue metabolites (initially discovered by high-resolution in vitro techniques on cancer specimens) can be translated into in vivo protocols based on noninvasive magnetic resonance spectroscopy (MRS). Using 1 H- and 31 P-MRS on living animals or patients, a decrease in citrate and polyamines in prostate cancer, an increase of cholines in breast cancer, as well as a decreased NAA and an increased lactate in gliomas during cancer progression can be assessed noninvasively. MRS can be used to follow up conventional cytotoxic as well as targeted anticancer therapies, which has been extensively done in animal models of cancer. This review focuses on applications and protocol development for in vivo 1 H- and 31 P-MRS on small animal models as well as on larger animals in cancer research, diagnosis, and treatment.