In vivo glucose sensing and regulation is necessary for patients with carbohydrate metabolism disorders, particularly those caused by diabetes mellitus. Self‐monitoring of blood glucose is part of the daily routine for such patients, and the measurement of capillary blood glucose, using different enzymatic assays in combination with photometric or electrochemical detection, still remains the standard method. Research activities concentrate on developing minimally invasive and noninvasive methodologies, the latter being based on spectroscopic techniques exploiting optical glucose specific characteristics, i.e. wavelength‐dependent absorptivities and refractive indices, mainly in the near‐infrared (NIR), or the rotation of linearly polarized radiation in the visible range. Owing to the complexity of the integrally probed tissue, and the presence of many interfering compounds, only multivariate spectroscopic measurement strategies, using several wavelengths, can be used for a noninvasive in vivo assay of glucose. Further complications arise from the heterogeneous distribution of glucose in the intravascular, interstitial and intracellular space and differences in their dynamics. In contrast to the established reference methodology which uses capillary blood, especially for the hypoglycemic concentration range, the imprecision observed so far within several experimental optical approaches is regarded as unacceptable for patient self‐monitoring. Further research is needed to establish an in vivo glucose assay for diabetic patients, or for direct monitoring in intensive care units and operating theatres, based on optical spectroscopy.