One major challenge for in vitro genotoxicology is the determination of the genotoxic mode of action of tested compounds. The quantification of the phosphorylation of the histones H3 (pH3) and H2AX (γH2AX) allows an efficient discrimination between aneugenic and clastogenic compounds. However, these two biomarkers do not permit to deduct the specific mechanisms involved in the action of clastogenic compounds. The aim of this study was to investigate other possible cellular biomarkers allowing differentiating clastogenic properties. For this purpose, we analyzed γH2AX and pH3 plus six other biomarkers involved in the DNA damage signaling pathway in HepG2 cells treated with nine clastogens exhibiting different mechanisms of action, as well as one aneugen. All compounds were tested at various concentrations and with kinetics of 2, 6, 24 and 48 hr. Our results demonstrate the activation of the investigated biomarkers by the tested compounds in a time and concentration dependent manner. Notably, we observed for some nondirect genotoxic clastogens, notably dNTPs pool imbalance inducers, a different kinetic of DNA damage induction compared with direct genotoxins (oxidative stress). However, no specific biomarker signature of mechanisms of clastogenic action could be specified. Multiparametric analysis demonstrates a strong correlation between γH2AX and p-p53(S15) for clastogen compounds.