The intracellular aqueous volumes (Vin) of microorganisms are fundamental data that can be used for estimating absolute cellular enzyme and metabolite concentrations. Because traditional methods are time-consuming and costly, the Vin data have been largely estimated ambiguously. Here we developed an NAD+ concentration-dependent method and demonstrated its usefulness for accurate estimation of the Vin value of Escherichia coli cells. The Vin value of E. coli BL21(DE3) cells was determined to be 1.9μL·mg-1, which is 17% lower than that of the commonly assumed data. Similarly, the Vin value of Agrobacterium tumefaciens AGL1 cells was determined to be 1.8μL·mg-1. Because NAD+ is routinely quantified during metabolite analysis, it may be integrated into metabolomic data collection with little additional time and labor expenditure. This method should also be applicable to estimate the Vin data of other prokaryotic microorganisms.