Immobilized-enzyme reactors (IMERs) are flow-through devices containing enzymes that are physically confined or localized with retention of their catalytic activities. IMERs can be used repeatedly and continuously and have been applied for (bio)polymer degradation, proteomics, biomarker discovery, inhibitor screening, and detection. Online integration of IMERs with analytical instrumentation, such as high-performance liquid chromatography (HPLC) systems, reduces the time needed for multi-step workflows, reduces the need for sample handling, and enables automation. However, online integration can also be challenging, as reaching its full potential requires complex instrumental setups and experienced users. This review aims to provide an assessment of recent advances and challenges in online IMER-based (analytical) LC platforms, covering publications from 2014-2021. A critical discussion of challenges often encountered in IMER fabrication, sample preparation, integration into the analytical workflow, long-term usage, and of potential ways to overcome these is provided. Finally, the obstacles preventing the proliferation of IMERs as efficient tools for high-throughput pharmacological, industrial, and biological studies are discussed
