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[Abstract] Intermediates of tetrapyrrole biosynthetic pathway are low-abundant compounds, and their quantification is usually difficult, time consuming and requires large amounts of input material. Here, we describe a method allowing fast and accurate quantification of almost all intermediates of the heme and chlorophyll biosynthesis, including mono-vinyl and di-vinyl forms of (proto) chlorophyllide, using just a few millilitres of the cyanobacterial culture. Extracted precursors are separated by High Performance Liquid Chromatography system (HPLC) and detected by two ultra-sensitive fluorescence detectors set to different wavelengths.