Vaccines are beginning to be explored for as measures to prevent cancer. Since determining the efficacy of vaccines by evaluating disease outcome requires a long time, there is an urgent need for early predictive biomarkers. To this end, immunological endpoints that can be assessed weeks or months post-vaccination are currently being evaluated. However, when multiple vaccines are available, waiting for the development of humoral and cellular immunity could still cause delays, whereas early assessments would allow for a timely shift to more effective prevention modalities. Applying the phospho-flow technique to primary T cells, we examined the phosphorylation status of various proteins that shape the activation, proliferation, and differentiation of mucin 1 (MUC1)-specific CD4+ T cells within the first 24 hours post-immunization. It is known that a vaccine composed of a MUC1-derived peptide loaded on dendritic cells is more effective in eliciting T-cell responses than a vaccine including the same peptide plus an adjuvant. Both these vaccines stimulate T cells more effectively in wild-type (WT) than in MUC1-transgenic mice. We examined if the signaling events downstream of the TCR or linked to various proliferative and survival pathways, monitored in two different hosts as early as 3, 6, 12 and 24 hours post-immunization, could predict the differential potential of these two MUC1-targeting vaccines. The signaling signatures that we obtained primarily reflect differences between the vaccines rather than between the hosts. We demonstrate the feasibility of using a phospho-flow-based approach to evaluate the potential of a given vaccine to elicit a desired immune response.