Targeted proteomics is a mass spectrometry-based protein quantification technique with high sensitivity, quantitative accuracy and reproducibly. As a key component in the multi-omics toolbox of systems biology, targeted liquid chromatography-selected reaction monitoring (LC-SRM) measurements are critical for enzyme and pathway identification and design in metabolic engineering. To fulfill the increasing need for analyzing large scale samples with faster turnaround time in systems biology, high-throughput LC-SRM is greatly needed. Even though nanoflow LC-SRM has better sensitivity, it lacks the speed offered by microflow LC-SRM. The recent advancements of mass spectrometry instrumentation significantly enhance the scan speed and sensitivity of LC-SRM thereby creating opportunities for applying the fast speed of microflow LC-SRM without losing peptide multiplexing power nor sacrificing much sensitivity. Here, we studied the performance of microflow LC-SRM versus nanoflow LC-SRM with 339 peptides representing 132 enzymes in Pseudomonas putida KT2440 grown on various carbon sources. The results from the two LC-SRM platforms are highly correlated. In addition, the response curve study of 248 peptides demonstrates that microflow LC-SRM has comparable sensitivity for majority of detected peptides and better mass spectrometry signal and chromatography stability than nanoflow LC-SRM.