Understanding and predicting whether new drug candidates will be safe in the clinic is a critical hurdle in pharmaceutical development, that relies in part on absorption, distribution, metabolism, excretion (ADME) and toxicology studies in vivo. Through toxicity and absorption analyses of a number of drugs, we find that zebrafish is generally predictive of drug toxicity although liver microsome assays reveal specific differences in metabolism of compounds between human and zebrafish livers, likely resulting from the divergence of the Cytochrome P450 superfamily between species. To reflect human metabolism more accurately, we generated a transgenic “humanized” zebrafish line that expresses the major human Phase I detoxifying enzyme, CYP3A4, in the liver. Here, we show that this humanized line shows an elevated metabolism of CYP3A4 specific substrates compared to wild-type zebrafish. The generation of this first described humanized zebrafish liver, suggests such approaches can enhance the accuracy of the zebrafish model for toxicity prediction. We subsequently utilise transcriptomics to identify highly upregulated genes as biomarkers of toxic responses in this model, finding candidate biomarkers which recurred in multiple treatments. Through promoter isolation and fosmid recombineering, eGFP reporter transgenic zebrafish lines were generated and showed a dose and time dependent induction in endodermal organs to reference drugs and an expanded drug set. Thus through integrated transcriptomics and transgenic approaches, we have developed a humanised zebrafish toxicity assay coupled with parallel independent zebrafish in vivo screening platforms able to predict organ toxicities of preclinical drugs.