Gut content analyses of fish are typically conducted using methods based on visual identification. These can lead to inconsistent results because of their subjective nature, especially when sample sizes are limited or food items are not resolvable. New approaches are required to increase the accuracy of gut content analysis. We investigate whether thermochemolysis with TMAH and GC-MS detection can qualitatively and quantitatively analyse the gut contents of two Monacanthid fishes fed seagrass, epiflora and epifauna under controlled conditions. The three food items could be readily differentiated when analysed individually, seagrass could be distinguished from epiphytes (epifauna + epiflora) in the gut, and quantitative data could be obtained using marker compounds unique to a food item. Thermochemolysis with TMAH and GC-MS represents a new technique for the gut content analysis of fish which can complement traditional techniques and be applied to samples that are very small (0.5-2 mg) and contain difficult to separate items.