Salmonella enterica serovar Pullorum affecting poultry causes pullorum disease and results in severe economic loss in the poultry industry. Currently, it remains a major threat in countries with poor poultry surveillance and no efficient control measures. As S. Pullorum could induce strong humoral immune responses, we applied an immunoscreening technique, the in vivo-induced antigen technology (IVIAT), to identify immunogenic bacterial proteins expressed or upregulated during S. Pullorum infection. Convalescent-phase sera from chickens infected with S. Pullorum were pooled, adsorbed against antigens expressed in vitro, and used to screen an S. Pullorum genomic expression library. Forty-five proteins were screened out, and their functions were implicated in molecular biosynthesis and degradation, transport, metabolism, regulation, cell wall synthesis and antibiotic resistance, environmental adaptation, or putative functions. In addition, 11 of these 45 genes were assessed for their differential expression by quantitative real-time reverse transcription-PCR (RT-PCR), revealing that 9 of 11 genes were upregulated to different degrees under in vivo conditions, especially the regulator of virulence determinants, phoQ. Then, four in vivo-induced proteins (ShdA, PhoQ, Cse3, and PbpC) were tested for their immunoreactivity in 28 clinical serum samples from chickens infected with S. Pullorum. The rate of detection of antibodies against ShdA reached 82% and was the highest among these proteins. ShdA is a host colonization factor known to be upregulated in vivo and related to the persistence of S. Typhimurium in the intestine. Furthermore, these antigens identified by IVIAT warrant further evaluation for their contributions to pathogenesis, and more potential roles, such as diagnostic, therapeutic, and preventive uses, need to be developed in future studies.