Liquid chromatography tandem mass spectrometry (LC–MS/MS) provides a powerful means to analyze intracellular metabolism. A prerequisite to accurate metabolomics analysis using LC–MS/MS is a robust sampling and extraction protocol. One unaddressed area in sampling is a detailed examination of a suitable method for anaerobic cultures grown in complex media. Given that a vast majority of bacteria are facultative or obligate anaerobes that grow to low biomass density and need to be cultured in complex media, a suitable sampling and extraction strategy for anaerobic cultures is needed. In this work, we develop a fast-filtration method using pressure-driven Swinnex® filters. We show that the method is fast enough to provide an accurate snapshot of intracellular metabolism, reduces matrix interference from the media to improve the number of compounds that can be detected, and is applicable to anaerobic and aerobic liquid cultures grown in a variety of culturing systems. Furthermore, we apply the fast filtration method to investigate differences in the absolute intracellular metabolite levels of anaerobic cultures grown in minimal and complex media.