© 2019, © 2019 Informa UK Limited, trading as Taylor & Francis Group. Methods for nanoparticles preparation often employ organic solvents in order to solubilize the non-polar constituents of the final nanostructures. In the research process, nanoparticles are assayed as aqueous suspensions in several cases, so that an excessive residual concentration of the organic solvent needs to be avoided since may lead to undesired secondary effects during biological tests. Despite the importance, residual solvent concentration is rarely determined, making necessary the development of quantification methods suitable for this purpose. Acetone is frequently used in drug delivery systems preparation, being capable to exert significant toxicities both, in vitro and in vivo. Thus, a simple and inexpensive UV–Vis spectrophotometric method is proposed to directly determine acetone from nanoparticles suspensions employing its reaction with vanillin. Central composite designs were employed to correct and optimize the quantification method, which was then validated according to international guidelines. The optimized method resulted accurate, precise, and linear in the range of 10–50 µg/mL, with an R2 of 0.998 and limits of detection and quantification of 2.6 and 7.8 µg/mL, respectively. The effect of several surfactants employed during nanoparticles preparation was not detrimental to the method. The proposed procedure can be successfully applied to directly quantify acetone from nanoparticles suspensions.