Paralichthys adspersus (Steindachner, 1867) is a flounder distributed in Peru and Chile, with importance in the Peruvian aquaculture but with variable growth performance of organisms during larval development and early juvenile stage. Molecular techniques, like transcriptomic analysis, have facilitated the identification of several expressed genes useful for understanding organism response under captivity. For evaluating growth during P. adspersus larvae development, we tested the amplification efficiency of ten primer pairs designed from RNA-Seq data previously generated from liver and muscle of juveniles with fast- and slow-growth. Primers for amplification of elongation factor 1-alpha (Ef1a1) and 40s ribosomal protein (40S) genes, were evaluated as potential candidate endogenous reference markers. Also, we included tests for IGF-BP and b-Actin, as target and reference genes, respectively. Ten-fold serial dilutions were prepared from cDNAs of organisms during 60 and 90 days post hatch. Standard curves were evaluated for each marker, in triplicates, by plotting the template quantity against threshold cycle (Ct). Efficiency (E) was calculated by the Pfaff method. Ct values were from 17 ± 0.02 to 32 ± 0.70, in approximately 7 ng template. E-values were calculated from 80% to 105%, with a correlation index (R squared) of 0.96 to 0.99. To validated specificity of primers, all PCR products were evaluated through Dissociation Curve Analysis and agarose gels, with an expected single peak and band of the expected size, respectively. Finally, alignment of sequenced PCR products to transcripts confirmed the genes amplified. These results demonstrated the potential of these marker primers designed from juvenile transcriptomes, for the relative quantification of genes expression during different larval stage samples.