There is an urgent demand for more efficient and ethical approaches in chemical risk assessment. Using 17a-ethinylestradiol (EE2) as a model compound, this study established a live-animal alternative embryo benchmark-dose (BMD) assay for rainbow trout (RBT Oncorhynchus mykiss) to derive transcriptomic points-of-departure (tPODs). Embryos were exposed to graded concentrations of EE2 (0, 1.13, 1.57, 6.22, 16.3, 55.1, and 169 ng/L) from hatch to 4 and up to 60 days post-hatch (dph) to assess molecular and apical responses, respectively. Whole proteome analyses of alevins did not show clear estrogenic effects, while transcriptomics revealed responses that were in agreement with apical effects including excessive accumulation of intravascular and hepatic proteinaceous fluid and significant increases in mortality at 55.1 and 169 ng/L EE2 at later time points. Transcriptomic BMD analysis estimated the median of the 20th lowest geneBMD to be 0.18 ng/L the most sensitive tPOD. Other estimates (0.78, 3.64, and 1.63 ng/L for the tenth percentile geneBMD, first peak geneBMD distribution, and median geneBMD of the most sensitive overrepresented pathway, respectively) were within the same order of magnitude as empirically derived apical P ODs for EE2 in the literature. This 4-day alternative RBT embryonic assay was effective in deriving protective tPODs for EE2. Overall design: mRNA profiles of whole rainbow trout alevins (Oncorhynchus mykiss 4 days post hatch) exposed to solvent control (0.01% DMSO), 1.13, 1.57, 6.22, 16.3, 55.1, and 169 ng/L 17a-ethinylestradiol (measured concentrations) from hatch to 4 days post-hatch. Three replicates of pooled alevins (3 alevins per replicate) per treatment group.