The heme biosynthetic pathway is a tightly regulated metabolic network essential for cellular function. Disruptions in this pathway can lead to the accumulation of intermediates, which is a hallmark of porphyria and other metabolic disorders and to iron metabolism imbalance. Porphyrin accumulation serves as a key diagnostic marker for porphyria and is crucial for understanding its pathophysiology. In this study, we employ NMR spectroscopy and enzymatic assays to investigate the inhibitory effects of accumulated metabolites on key enzymes within the pathway. We demonstrate several critical examples, including the inhibition of coproporphyrinogen oxidase by d-aminolevulinic acid and the inhibitory effects of various metabolites and by-products on uroporphyrinogen III synthase and porphobilinogen deaminase. Furthermore, fluxomic analyses are used to track and quantify the accumulation of different by-products, providing a comprehensive view of metabolic perturbations. Our results emphasize the importance of porphyrin accumulation in disease diagnosis and highlight potential therapeutic strategies to mitigate the pathological consequences of metabolic imbalances in the heme biosynthetic pathway.