Lignin content of hydrothermal pretreated bamboo chips was determined by the two methods: TAPPI standard method (222om-06) and TAPPI standard method without benzene-alcohol extraction (BAE). The results showed that including BAE resulted in lower Klason lignin (ML) and acid soluble lignin (ASL) measurements in the prehydrolyzed substrate, that is to say, BAE removed parts of KL and ASL. Therefore, the TAPPI standard method should be modified by omitting the BAE for lignin measurements of pretreated substrate. The following lignin removal analysis suggested that lignin was removed from the bamboo substrate during pretreatment by a combination of degradation reaction and deconstruction; thereafter the pseudo lignin generated in the hydrothermal pretreatment and condensation reaction between the lignin fragments accounted for the later la increase. (C) 2013 Elsevier Ltd. All rights reserved.