Recent developments in multiplex technologies enable the determination of a large nu\mber of soluble proteins such as cytokines in various biological samples. More than a one-by-one determination of the concentration of immune mediators, they permit the establishment of secretion profiles for a more accurate description of conditions related to infectious diseases or vaccination. Cytokine profiling has recently been made available for bovine species with the development of a Luminex® technology-based 15-plex assay. Independently from the manufacturer, we evaluated the bovine cytokine/chemokine multiplex assay for limits of detection, recovery rate, and reproducibility. Furthermore, we assessed cytokine secretion in blood samples from 107 cows upon stimulation with heat-killed bacteria and TLR2/4 ligands compared to a null condition. Secretion patterns were analyzed either using the absolute concentration of cytokines or using their relative concentration with respect to the overall secretion level induced by each stimulus. Using Partial Least Square-Discriminant Analysis, we show that the 15-cytokine profile is different under Escherichia coli, Staphylococcus aureus, and Streptococcus uberis conditions, and that IFN-γ, IL-1β, and TNF-α contribute the most to differentiate these conditions. LPS and E. coli induced largely overlapping biological responses, but S. aureus and S. uberis were associated with distinct cytokine profiles than their respective TLR ligands. Finally, results based on adjusted or absolute cytokine levels yielded similar discriminative power, but led to different stimuli-related signatures.