Serotonin and kynurenine are formed following metabolism of the essential amino acid tryptophan. Both molecules play important biological roles and the balance of how tryptophan metabolism varies to either the serotonin or kynurenine pathway may provide key insight into the inflammatory status of the biological region. At present complex chromatographic methods are utilised which predominately focus on either monitoring analytes in the serotonin or kynurenine pathway rather than both. Our study develops a simple yet robust methodology for the monitoring of tryptophan metabolism. We utilised isocratic reverse phase high-performance liquid chromatography with simultaneously dual electrochemical detection. This approach allowed for separation of co-eluted analytes and identification of analytes from both pathways within 14 minutes. For all analytes, limits of detection were <35 nM. No crosstalk was observed when dual simultaneous detection was conducted in a radial flow cell. Responses from the hippocampus, blood and ileum mucosa highlighted that each region had a varying ratio of serotonin to kynurenine pathway, indicating varied approaches to tryptophan metabolism. The developed method can monitor how the metabolism of tryptophan varies between the two pathways which can provide insight into the inflammatory state of reach region with age and disease.