The intense but transient increase in nuclear magnetic resonance signal provided by dynamic nuclear polarization (DNP) has enabled a range of new applications, including rapid, minimally-invasive metabolic imaging at the second timescale. This chapter provides an overview of the hyperpolarized carbon-13 labeled agents prepared by dissolution-DNP used in vivo and their biochemical interactions, categorized by metabolic pathway. The required characteristics of a suitable probe and an efficiently-polarizing formulation are described, as are practical considerations for formulation development and metabolite identification. Relevant biochemical principles to aid in the interpretation of the apparent metabolism of the probes are introduced with illustrative examples.