Description:We examined, for the first time using untargeted and targeted metabolomics, the effect of several commonly-used methods of anesthesia and euthanasia for collection of skeletal muscle, liver, heart, adipose and serum of C57BL/6J mice. The data revealed tissue-specific impacts of different anesthesia and euthanasia strategies. Based on these findings, we present a more optimal collection strategy mammalian tissues and recommend that rodent tissues intended for metabolomics studies be collected under anesthesia rather than post-euthanasia.