AbstractAlkaline pre-extraction and hydrothermal hydrolysis were carried out with eucalyptus chips. Two methods were used to determine and calculate the extraction yield of five-carbon sugars. One is an indirect method, based on the difference of pentosan content between the chips before and after pre-extraction. The other method is to directly measure the content of five-carbon sugars in the extracting solution. The results indicated that there was a defined relationship between the two methods. For alkaline pre-extraction, a good logarithmic relationship was shown. There was a good linear relationship for hot water prehydrolysis. So the pentosan content of the extracted chips could be predicted from the results of the latter method, referring to the two relational expressions. In this study, a simple and rapid method of spectrophotometry was introduced, which will help in the evaluation of extraction yield of five-carbon sugars during biomass processing.