The description and identification of metabolites using Gas Chromatography - Mass Spectrometry (GC-MS) is a powerful tool to study the cnidarian-dinoflagellate symbiosis. The applications range from first descriptions of metabolite profiles to the identification of specific metabolic responses (e.g. biomarkers) of the cnidarian metaorganism (\u2018holobiont\u2019), including its associated symbionts, under stress.The following step-by-step protocol is optimized to characterize the carbohydrate fraction from Aiptasia anemones or coral fragments, as well as their algal symbionts, using a targeted GC-MS approach. The protocol was previously used in Ochsenk\u00fchn et al. (2017) where it identified the following carbohydrates (floridoside, inositol, mannitol, glucose, glycerol, galactose, ribose and fructose) as well as some amino acids (glycine, alanine, valine and proline).In addition to the step-by-step protocol for sample preparation and derivatization, we provide detailed settings for the Agilent GC-MS system (GC (Agilent 7890A) and MS (Agilent 5975C)) in a separate document.