In contrast to the abundance of systems-oriented approaches describing changes on the transcriptome or proteome level, relatively few studies have employed the metabolome. The goal of the presented research was to identify as many intracellular metabolites as possible in a Spiroplasma melliferum extract by flow injection time-of-flight mass spectrometry. The Mollicutes class bacterium S. melliferum is a member of a unique category of bacteria that have in common the absence of a cell wall, a reduced genome, and simplified metabolic pathways. Metabolite identification was confirmed by fragmentation of previously detected ions by target mass spectrometry. The selected liquid chromatography approach, hydrophilic interaction chromatography with amino and silica columns, effectively separates highly polar cellular metabolites prior to their detection on a high accuracy mass spectrometer in positive and negative acquisition mode for each column. Here we present reliable measurement of 76 metabolites, including components of sugar, amino acid, and nucleotide metabolism. We have identified about a third of the possible intracellular S. melliferum metabolites predicted by genome annotation.