We present a method to perform absolute quantification of glycogen in human embryonic stem cells (hESCs) in situ based on the use of Raman microspectroscopy. The proposed quantification method was validated by comparison to a commonly used commercial glycogen assay kit. With Raman microspectroscopy, we could obtain the glycogen content of hESCs faster and apparently more accurately than with the kit. In addition, glycogen distributions across a colony could be obtained. Raman spectroscopy can provide reliable estimates of the in situ glycogen content in hESCs, and this approach should also be extensible to their other biochemical constituents as well as to other cell types.