Valorization of lignocellulosic biomass into biofuels has received considerable attention for decades with tremendous thermochemical pretreatments proposed. However, inconsistencies among studies in lignin removal can often be misleading and even violate the chemical mechanism, which might be attributed to the variation in the filtration method used after pretreatment. A phenomenon, promoting pretreatment strategies based on sugar conversion efficiency or yield only from single and random lowsolid enzymatic hydrolysis, was also observed. Herein, the effects of filtration methods using a slow drip metal filter (SDMF) with an aperture of 0.2 mm and Whatman grade 4 filter paper (WGFP) with an aperture of 20-25 mu m under vacuum filtration after liquid hot water (LHW), acid, and alkali pretreatments on the chemical composition of the solid and liquid, sugar recoveries, and lignin removal and solid loading (2-15%) on enzymatic hydrolysis of corn stover and poplar wood were investigated. Results showed that SDMF increased the glucan content (1.1-3.0%) in pretreated biomass and induced solid loss (3.8-12.6%) into the filtrates, resulting in lower sugar recoveries and overestimated lignin removal compared to WGFP. 5-Hydroxymethylfurfural (HMF) and furfural derived from sugar degradation triggered pseudolignin formation, inducing a negative lignin removal for both LHW and acid pretreated biomass. In comparison, alkali pretreatment neutralized the weak acids released from sugar decomposition and achieved a robust delignification (67.8-90.8%). Solid loading used for enzymatic hydrolysis of pretreated biomass determined sugar concentration, conversion efficiency, and yield. This result indicates that promoting pretreatment technologies based on sugar conversion efficiency or yield of enzymatic hydrolysis at low solid loading may be misleading. This work demonstrates that the filtration method used for solid and liquid separations after pretreatment and solid loading for enzymatic hydrolysis should be cautiously selected and implemented when valorizing biomass into biofuels.