Evaluation of the immune status must be assessed in highly controlled conditions to be amenable for reproducibility. Furthermore, whereas the cellular response plays a major role, immunity has been measured for a long time through antibody production. For the purpose of assessing immune cell responsiveness in ruminants, we developed and validated a whole blood assay coupled to a high-throughput multiplex cytokine assay using the Luminex technology. A custom Milliplex assay was developed with MERCKMillipore company for the measurement of 15 bovine/ovine cytokines of both the innate and adaptive immunity, and chemokines. Whole blood samples were collected from 110 dairy cows in an INRA experimental unit, and immediately stimulated during 24 hours with heat-killed bacteria (Escherichia coli, Staphylococcus aureus, and Streptococcus uberis), Toll-like receptor ligands (FSL-1 for TLR2, LPS for TLR4, and Gardiquimod for TLR7-8), or soluble anti-CD3/CD28 monoclonal antibodies. Cytokine secretion was determined and compared with a sample prepared in the same condition but without any stimulant. Using ANOVA and data mining methods (PCA, clustering…), we showed that the protocol is able to detect differences between bacteria, with S. uberis being the most potent to induce a response, compared to S. aureus and E. coli that were distinct but more closely related to each other. Similarly, TLR ligands could be distinguished, and Gardiquimod was significantly different from the MyD88- associated TLR2/4-activating ligands. LPS and E. coli provided very similar response profiles confirming previous data indicating that a large part of the E. coli response is mediated through LPS signaling. A high variability of response was detected amongst cattle suggesting environmental and genetic variations of the cytokine response. Altogether, results indicate that cytokine profiling in cattle is achievable and can be used in further work to delineate more precisely the responsiveness of cattle in various situations, including variability of the genetic background.