The cattle tick Rhipicephalus (Boophilus) microplus is one of the most harmful ectoparasites affecting bovines worldwide. It represents a major threat to livestock industry due to the economic losses caused and diseases associated with these ticks. The most important tick control strategy has been the use of ixodicides, resulting in chemically resistant tick populations. It is necessary to understand the mechanisms that result in resistance so as to create new strategies increasing the lifespan of ixodicides or finding alternative targets to produce new acaricides. In this paper, in order to obtain an insight into the mechanisms that govern ixodicides resistance, we will compare the hemolymph proteome of two tick R. microplus strains, one susceptible (MJ) and one resistant (SA) to ixodicides, using HPLC and 2D electrophoresis. Significant differences were found in protein content between strains using HPLC. 2D electrophoresis revealed that 68 hemolymph protein spots were common between strains; however, 26 spots were unique to the susceptible strain MJ and 5 to the resistant strain SA. The most distinctive protein spots on the preparative gels were selected for further analyses. Nine protein spots were identified by mass fingerprinting, revealing proteins that may have a role in the ixodicides resistance or susceptibility. In this paper, we present the tick hemolymph proteome revealing a set of proteins which suggest a possible role in tick detoxification.