Recently, a bacterial second messenger, guanosine 5'-diphosphate 3'-diphosphate (ppGpp), has been detected in chloroplasts. However, because ppGpp concentration in plants is much lower than that in bacteria, detailed analysis of ppGpp in plants has not been performed. A highly sensitive quantification method is required for further characterization of ppGpp function in chloroplasts. Here, we report a new method that allows for the highly sensitive and selective high-throughput quantification of ppGpp by ultra-performance liquid chromatography (UPLC) coupled with a tandem quadrupole mass spectrometer (qMS/MS) equipped with an electrospray interface (ESI). This method requires only ~100 mg of plant tissue for ppGpp quantification. We used this method to measure ppGpp levels in Arabidopsis thaliana under different light conditions. A. thaliana accumulated ppGpp during dark periods. This method will be helpful to further characterize the stringent response in higher plants.