Abstract Lignocellulose, the most abundant and renewable resource on Earth is an important raw material, which can be converted into high value products. However, to this end, it needs to be pretreated physically, chemically, or biologically. Its complex structure and recalcitrance against physical, chemical, or biological degradation render its breakdown an important target of study. The understanding of the enzymatic processes of lignocellulose breakdown and the changes in its chemistry are thus essential. Here, we review the current analytical challenges in the analysis of lignocellulose composition, lignocelluloytic pretreatment, analysis of enzymatic hydrolysis catalyzed by cellulases or hemicellulases and their biotechnological applications. Complex techniques including biochemical, genomic, and metagenomics methods such as high performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD), Respiration Activity Monitoring System (RAMOS), and next-generation sequencing are described. HPAEC-PAD is a promising, rapid, and reliable analytical technique for sugar quantification following lignocellulose breakdown. RAMOS is an effective technique for monitoring the growth of microorganisms during the different phases of enzyme production, enzymatic hydrolysis, and fermentation. The emergence of high throughput, next-generation sequencing techniques has enriched the databases of genes encoding glycoside hydrolase classes commonly involved in lignocellulose decomposition, and this knowledge can be readily used to analyse the involved processes. Still, novel analytical methods are highly welcome to understand the complete process of lignocelluloytic breakdown. In order to decrease environmental pollution and to save energy, lignocellulose conversion needs to be promoted in order to effectively compete with fossil resources on a global scale in future.