Partial hepatectomy (PHx) is a frequently used experimental model for the study of liver regeneration. Real-time quantitative PCR (qPCR) has become the one of the methods of choice for expression profiling of selected genes in order to elucidate the regulation of liver function and regeneration. The expression of five commonly used housekeeping genes (HKGs; Alb, UBC, Hprt, Ywhaz, and GAPDH) were evaluated by qPCR in 70% and 90% rat PHx model at 1, 2, and 7 d after PHx. We set up a closely controlled qPCR procedure validating each critical step and the gene expression stability was statistically evaluated by linear regression and analysis of variance. Our results showed the HKG best suited for the evaluation of gene expression in the extended 90% PHx model is Hprt. The amplification of an HKG can be omitted when the same amount of cDNA from all samples is introduced into the amplification reaction. Determination of cDNA concentration employing the bioanalyzer proved to be an easy and reproducible approach. Using this technique the potential regulation of the transcription level of the HKG in response to the experimental condition tested or the stability of a housekeeping gene becomes irrelevant.