Having the ability to monitor metabolic activity at the scale of single bacterial cells non-invasively would enable us to follow changes in the distribution of activity in bacterial systems which is of major importance for topics such as integration of metabolism and development, metabolic engineering, microbial activity and drug resistance, cell-cell interactions, and quorum sensing. Here, we used laser tweezers Raman spectroscopy to monitor the in vivo real-time uptake and conversion of trehalose by single bacterial cells. This approach can be used for the quantitative determination of sugar uptake by a single bacterium and its metabolic response to the sugar application with time. We show that uptake of trehalose can be quantified in single living bacterial cells held in place by an optical trap while simultaneously collecting Raman spectra upon application of sugar to the medium. This technique yields real-time chemical information in a label-free manner, thus eliminating the limitations of toxicity of the isotopic probes common in studying these transport processes. It can substitute the laborious and time-consuming analytical evaluation. Although the single-cell Raman spectroscopy method demonstrated here is focused on the study of trehalose uptake by Sinorhizobium meliloti, the demonstrated approach is applicable to many different organisms and carbohydrates in general.