Natural Products (NP) are essential for the discovery of novel drugs and products for numerous biotechnological applications. The NP discovery process is expensive and time-consuming, having as major hurdles dereplication (early identification of known compounds) and structure elucidation, particularly the determination of the absolute configuration of metabolites with stereogenic centers. This review comprehensively focuses on recent technological and instrumental advances, highlighting the development of methods that alleviate these obstacles, paving the way for accelerating NP discovery towards biotechnological applications. Herein, we emphasize the most innovative high-throughput tools and methods for advancing bioactivity screening, NP chemical analysis, dereplication, metabolite profiling, metabolomics, genome sequencing and/or genomics approaches, databases, bioinformatics, chemoinformatics, and three-dimensional NP structure elucidation.