Nucleotides exemplify some of the building blocks of life, comprising DNA & RNA, participating in processes such as cell signaling and metabolism, and serving as carriers of metabolic energy. The quantification of these compounds in biological samples is critical for researchers to understand complex systems. Herein, we demonstrate an anion exchange chromatography method utilizing a pH range of 8 to 10, which provides superior resolution and selectivity to previously reported methods and, more importantly, gives the flexibility to shift analyte selectivity if resolution between analytes is not optimal. We have applied the method to study the kinetics of the nucleotide pool in a bacterial cell-free lysate system that is producing RNA. Sample to sample runtimes are less than 18 min and recoveries greater than 96% were observed for all analytes through our methanol quench protocol with day-to-day variabilities less than 5%. This method reliably detects and quantifies all analytes that were expected to be observed in the process and helps lay the groundwork for future nucleotide research.