Cytotoxic T cells substantially contribute to the control of intracellular pathogens such as human immunodeficiency virus type 1 (HIV-1). Here, we evaluated the immunopeptidome of Jurkat cells infected with the vaccine candidate MVA.HIVconsv, which delivers HIV-1 conserved antigenic regions by using modified vaccinia virus Ankara (MVA). We employed liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify 6,358 unique peptides associated with the class I human leukocyte antigen (HLA), of which 98 peptides were derived from the MVA vector and 7 were derived from the HIVconsv immunogen. Human vaccine recipients responded to the peptide sequences identified by LC-MS/MS. Peptides derived from the conserved HIV-1 regions were readily detected as early as 1.5 h after MVA.HIVconsv infection. Four of the seven conserved peptides were monitored between 0 and 3.5 h of infection by using quantitative mass spectrometry (Q-MS), and their abundance in HLA class I associations reflected levels of the whole HIVconsv protein in the cell. While immunopeptides delivered by the incoming MVA vector proteins could be detected, all early HIVconsv-derived immunopeptides were likely synthesized de novo. MVA.HIVconsv infection generally altered the composition of HLA class I-associated human (self) peptides, but these changes corresponded only partially to changes in the whole cell host protein abundance.The vast changes in cellular antigen presentation after infection of cells with a vectored vaccine, as shown here for MVA.HIVconsv, highlight the complexity of factors that need to be considered for efficient antigen delivery and presentation. Identification and quantitation of HLA class I-associated peptides by Q-MS will not only find broad application in T-cell epitope discovery but also inform vaccine design and allow evaluation of efficient epitope presentation using different delivery strategies.