An important approach for developing a safer smallpox vaccine is to identify naturally processed immunogenic vaccinia-derived peptides rather than live whole vaccinia virus. We used two-dimensional liquid chromatography coupled to mass spectrometry to identify 116 vaccinia peptides, encoded by 61 open reading frames, from a B-cell line (homozygous for HLA class I A*0201, B*1501, and C*03) after infection with vaccinia virus (Dryvax). Importantly, 68 of these peptides are conserved in variola, providing insight into the peptides that induce protection against smallpox. Twenty-one of these 68 conserved peptides were 11 amino acids long or longer, outside of the range of most predictive algorithms. Thus, direct identification of naturally processed and presented HLA peptides gives important information not provided by current computational methods for identifying potential vaccinia epitopes.