4-hydroxyphenylpyruvate dioxygenase (HPD) is a key enzyme in the catabolism of tyrosine and therefore of great importance as a drug target to treat tyrosine-related inherited metabolic disorders (TIMD). Inhibition of this enzyme is therapeutically applied to prevent accumulation of toxic metabolites in TIMD patients. Nowadays an ex-herbicide, nitisinone, is used for this purpose and many more inhibitors are being explored and need to be tested. Here, we describe a colorimetric bacterial whole-cell screening system that allows quantifying the inhibitory effects of new human HPD inhibitors in a high-throughput and robust fashion. For this high-throughput screening (HTS) system we rely on the capability of recombinant E. coli that express human HPD, to generate a brown ochronotic pigment after the addition of tyrosine, whereafter this brown pigment can be quantified in a very specific and sensitive way by spectrophotometric analysis. Altogether, this robust and simple HTS screening system can be described as non-harmful, non-laborious and cost-effective with the aim to identify and evaluate novel therapeutic human HPD inhibitors for the treatment of TIMD.•This robust high-throughput screening system enables rapid identification and evaluation of potential inhibitors of human 4-hydroxyphenylpyruvate dioxygenase.•Simple and fast colorimetric quantification of the formation of ochronotic pigment.