Precise control of the level of protein expression in cells can yield quantitative and temporal information on the role of a given gene in normal cellular physiology and upon exposure to chemicals and drugs. This is particularly relevant to liver cells, in which the expression of many proteins, such as phase I and phase II drug metabolizing enzymes, vary widely between species, among individual humans, and upon exposure to xenobiotics. The most widely used gene regulatory system has been the tet-on/off approach. Although a 2^nd-generation tet-on transactivator was recently described, this not been widely investigated for its potential as a tool for regulating genes in cells and particularly in cells previously recalcitrant to the 1^st-generation tet-on approach such as hepatocyte-derived cells. Here, we demonstrate the development of two human (HepG2 and HuH7) and one mouse (Hepa1c1c7) hepatoma-derived cell lines incorporating a 2^nd-generation doxycycline-inducible gene expression system, and the application of the human lines to control the expression of different transgenes. The two human cell lines were tested for transient or stable inducibility of five transgenes relevant to liver biology, namely Phase I (CYP2E1) and Phase II (GSTP1) drug metabolism, and three transcription factors that respond to chemical stress (nuclear factor erythroid 2 p45-related factors 1 & 2 (NRF1 & NRF2) and NFKB1). High levels of functional expression were obtained in a time- and dose-dependent manner. Importantly, doxycycline did not cause obvious changes in the cellular proteome. In conclusion, we have generated hepatocyte-derived cell lines in which expression of genes is fully controllable.