Hyperpolarized 13C MRS is a powerful emerging technique for assessing metabolic liver diseases, including diabetes and fatty liver. Chemical shift separation allows monitoring of many metabolites at once. Here, we explore the possibility of co-polarizing [1-13C]pyruvate and [2-13C]dihydroxyacetone to simultaneously assess two important metabolic pathways that are altered in liver disease. Co-polarization was successful, with preserved T1 relaxation times. Although co-polarization led to modest decreases in polarization levels, all expected metabolites were observed when injected into a healthy rat. By examining two pathways at the same time, co-polarization can be an important tool for assessing liver disease.