Aims: The aims of the present work were to solubilize the abundantly expressed recombinant xylulokinase in Escherichia coli and to develop a reliable xylulokinase assay. Methods and Results: Three mutants of xylulokinase of Bacillus megaterium that were expressed at high level but formed insoluble protein in E. coli BL21(DE3)pLysS were selected for solubility study. The solubility of xylulokinase increased eight to 77-fold after introduction of molecular chaperones GroEL-GroES into the host. Conclusion: This investigation reports that GroEL-GroES minimizes the formation of insoluble protein in three highly expressed recombinant xylulokinases and an improved xylulokinase assay. Significance and Impact of the Study: Commercial production of bioethanol is critically dependent on the development of an efficient and low-cost process of enzymatic conversion of xylan, a major component in lignocellulose biomass, to xylulose-5-phosphate, which can then be channelled into pentose phosphate pathway and metabolized to ethanol. The improved intracellular xylulokinase activity is expected to facilitate the xylose degradation.