Quantification of nucleotides is an important part of metabolomics but has been hampered by the lack of fast, sensitive, and reliable methods. We present a less time-consuming, more sensitive, and more precise method for the quantitative determination of nucleoside triphosphates (NTPs), 5-ribosyl-1-pyrophosphate (PRPP), and inorganic pyrophosphate (PPi) in cell extracts. The method uses one-dimensional thin-layer chromatography (TLC) and radiolabeled biological samples. Nucleotides are resolved at the level of ionic charge in an optimized acidic ammonium formate and chloride solvent, permitting quantification of NTPs. The method is significantly simpler and faster than both current two-dimensional methods and high-performance liquid chromatography (HPLC)-based procedures, allowing a higher throughput while common sources of inaccuracies and technical problems are avoided. For determination of PPi, treatment with inorganic pyrophosphatase (PPase) of the radiolabeled phosphate is employed for removal of contaminating pyrophosphate. Biological examples performed in triplicates showed standard deviations of approximately 10% of the mean for the determined concentrations of NTPs.