Cyclic dinucleotides such as bis-(3',5')-cyclic dimeric adenosine monophosphate (c-di-AMP) and bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) represent important second messengers in bacteria. Although their synthesis has not been described in plants so far, they may be involved in the regulation of bacterial phytopathogen-plant interactions as well as rhizobium plant symbiosis. Here, we describe a sensitive and specific quantification method for c-di-AMP and c-di-GMP by HPLC-coupled tandem mass spectrometry. Additional linear dinucleotide metabolites and mononucleotides, as well as cyclic mononucleotides, can be simultaneously determined by this method.