The liver is among the most frequently targeted organs by noxious chemicals of diverse nature. Liver toxicity testing using laboratory animals not only raises serious ethical questions, but is also rather poorly predictive of human safety towards chemicals. Increasing attention is, therefore, being paid to the development of non-animal and human-based testing schemes, which rely to a great extent on in vitro methodology. The present paper proposes a rationalized tiered in vitro testing strategy to detect liver toxicity triggered by chemicals, in which the first tier is focused on assessing general cytotoxicity, while the second tier is aimed at identifying liver-specific toxicity as such. A state-of-the-art overview is provided of the most commonly used in vitro assays that can be used in both tiers. Advantages and disadvantages of each assay as well as overall practical considerations are discussed.