Enzymes play indispensable roles in producing biofuels, a sustainable and renewable source of transportation fuels. Lacking rational design rules, the development of industrially relevant enzyme catalysts relies heavily on high-throughput screening. However, few universal methods exist to rapidly characterize large-scale enzyme libraries. Therefore, assay development is necessary on an ad hoc basis to link enzyme properties to spectrophotometric signals and often requires the use of surrogate, optically active substrates. On the other hand, mass spectrometry (MS) performs label-free enzyme assays that utilize native substrates and is therefore generally applicable. But the analytical speed of MS is considered rate limiting, mainly due to the use of time-consuming chromatographic separation in traditional MS analysis. Thanks to new instrumentation and sample preparation methods, direct analyte introduction into a mass spectrometer without a prior chromatographic step can be achieved by laser, microfluidics, and acoustics, so that each sample can be analyzed within seconds. Here we review recent advances in MS platforms that improve the throughput of enzyme library screening and discuss how these advances can potentially facilitate biofuel research by providing high sensitivity, selectivity and quantitation that are difficult to obtain using traditional assays. We also highlight the limitations of current MS assays in studying biofuel-related enzymes and propose possible solutions.