High-throughput screening technologies have been lacking in comparison to the plethora of high-throughput genetic diversification techniques developed in biotechnology. This review explores the challenges and advancements in high-throughput screening for high-value natural products, focusing on the critical need to expand ligand targets for biosensors and increase the throughput of analytical techniques in screening microbial cell libraries for optimal strain performance. The engineering techniques to broaden the scope of ligands for biosensors, such as transcription factors, G protein–coupled receptors and riboswitches are discussed. On the other hand, integration of microfluidics with traditional analytical methods is explored, covering fluorescence-activated cell sorting, Raman-activated cell sorting and mass spectrometry, emphasising recent developments in maximising throughput.