Recently, sugars such as trehalose have been introduced into mammalian cells by overcoming the permeability barrier of cell membranes, and have provided improved tolerance against stresses associated with freezing and drying. However, the fate of the intracellular sugars has remained an open question. To address this issue, mouse oocytes were microinjected with 0.1 mol/l trehalose, and intracellular trehalose and glucose concentrations were determined during embryonic development using a high performance liquid chromatography and pulsed amperometric detection protocol. Trehalose was not detected in non-injected controls at any stage of development. In the microinjection group, the amount of intracellular trehalose progressively decreased as embryos developed. There was a corresponding increase in intracellular glucose concentration at the two-cell stage, suggesting cleavage of trehalose to two glucose molecules. In summary, this study presents a simple, highly sensitive protocol to determine intracellular sugars. The data reveal rapid elimination of microinjected trehalose during embryonic development. These findings have implications for designing osmolarity-optimized culture media for sugar-injected oocytes.