Lignocellulosic biomass is the future feedstock for the production of biofuel and bio-based chemicals. The pretreatment-hydrolysis product of biomass, so-called hydrolysate, contains not only fermentable sugars, but also compounds that inhibit its fermentability by microbes. To reduce the toxicity of hydrolysates as fermentation media, knowledge of the identity of inhibitors and their dynamics in hydrolysates need to be obtained. In the past decade, various studies have applied targeted metabolomics approaches to examine the composition of biomass hydrolysates. In these studies, analytical methods like HPLC, RP-HPLC, CE, GC-MS and LC-MS/MS were used to detect and quantify small carboxylic acids, furans and phenols. Through applying targeted metabolomics approaches, inhibitors were identified in hydrolysates and their dynamics in fermentation processes were monitored. However, to reveal the overall composition of different hydrolysates and to investigate its influence on hydrolysate fermentation performance, a non-targeted metabolomics study needs to be conducted. In this review, a non-targeted and generic metabolomics approach is introduced to explore inhibitor identification in biomass hydrolysates, and other similar metabolomics questions.