Background: The liver and spleen play a pivotal role in metabolism and immune response. During stress, neuroendocrine response induces changes in gene expression and its assessment demands the validation of the stability of the reference genes to perform relative gene expression experiments. Aim: The objective of this study was to determine the expression stability of four reference genes (GAPDH, ACTB, RNA18S, and HMBS) in the liver and spleen tissues from laying hens housed in conventional cage (CC) and cage-free (CF) egg production systems. Methods: Liver and spleen from Hy-Line Brown hens housed in CC and CF egg production systems were used. mRNA transcript levels were determined by qPCR and the gene expression stability was evaluated using geNorm, BestKeeper and NormFinder algorithms. Results: the most stable gene from liver tissue was ACTB in CC, CF and CC-CF group (overall data). In the spleen, the most stables genes were GAPDH (CC), HMBS (CF), and ACTB (CC-CF). Conclusion: the ACTB gene was the most stable gene in the liver, and GAPDH and HMBS genes were stable in spleen tissues that could be used for the normalization in qPCR experiments performed in liver and spleen tissues of laying hens housed CC and CF production systems.