The aim of this study was to adapt the comet assay in spermatozoa of the marine prawn Palaemon serratus to use it as a marker of sperm quality. Indeed, due to the characteristics of their spermatozoa, the measurement of DNA integrity is ones of the few markers, which can be transferred to crustaceans to assess the quality of their semen. In first time, the methods to collect and to maintain spermatozoa were optimized. The cell survival was estimated during kinetics of preservation (i.e. 1, 2, 4 and 8 h) in various suspension media to define the artificial seawater (ASW) as optimal. Several methods in the releasing of spermatozoa from the spermatophore of prawns were estimated with regard to their incidence both on the efficiency of extraction and the survival of cells. The pipetting up and down turned out to be the most successful and the least invasive technic. Secondly, the transfer of Comet assay was optimized by studying various times in both cell lysis (i.e. 1, 6, 18 h) and DNA denaturation (i.e. 15, 30 and 45 min), after in vitro exposure of spermatozoa toward a H2O2gradient as model genotoxicant. Results revealed that a minimum of 1 h in cell lysis and 15 min of DNA denaturation was sufficient to obtain valuable results, linked with a low compaction of DNA in spermatozoa of Palaemon sp. Finally, the sensitivity of P. serratus spermatozoa was assessed after in vitro exposures toward model genotoxicants displaying various modes of interaction with DNA (i.e. UV-C, 5-30 sec; H2O2, 5-10 μM and MMS, 0.5-5 mM) and some environmental contaminants known or suspected to be genotoxic (i.e. cadmium and diuron, 0.015-1.5 μg.L-1; carbamazepine, 0.1-10 μg.L-1) toward invertebrates. The low variability of the baseline level of DNA strand breaks recorded in controls highlighted the robustness of the method. P. serratus spermatozoa displayed significant DNA damage from the lowest doses tested for all model genotoxicants, but conversely, no genotoxic effect of tested environmental contaminants was observed. These results could suggest a difference in the response or sensitivity of spermatozoa to environmental genotoxicity between invertebrate species, and therefore the interest of Palaemonidae prawns in ecogenotoxicology. In conclusion, the present study underlines the potential of the Comet assay as a marker to assess the contamination impact on the sperm quality in Palaemonidae prawns in view to a potential application for in situ biomonitoring surveys.