Objective To identify a cost-efficient alternative antibody testing strategy for screening and confirmation of HIV infection by rapid simple tests (RSTs) and enzyme-linked immunosorbent assays (ELISAs). Methods Four RSTs (RST1,RST2, RST3, and RST4 ) and five ELISAs (ELISA1, ELISA2, ELISA3, ELISA4, and ELISA5) were evaluated in two phases by using banked and serum specimens prospectively collected at regional hospitals and voluntary counseling and testing (VCT)centers in Beijing. A total of 200 banked serum specimens were included in the first phase, including 62 HIV-positive, 127 HIV-negative and 11 indeterminate specimens. All specimens were tested by four RSTs and five ELISAs respectively. The second phase involved prospective testing of 389 routine specimens, including 92 HIV-positive, 287 HIV-negative, and 10 indeterminate specimens. All the specimens were tested by two RSTs (RST2 and RST4) and three ELISAs (ELISA1, ELISA3,and ELISA4), which were selected for their respective excellent sensitivity and/or specificity. Western blot (WB) was used as a gold standard for confirming the reactivity of all the specimens. Results Sensitivity, specificity, and efficacy were calculated for each assay in two phases. In the first phase, four assays (ELISA4, RST2, RST3, and RST4) had a specificity of 100%. For the determination of efficacy, ELISA4, RST2, and RST4 were selected in the second phase. ELISA1 and ELISA3 which have a sensitivity of 95.9% and 93.2% respectively also entered this phase. In the second phase, all the five assays (ELISA1, ELISA3, ELISA4, RST2, and RST4) had a sensitivity and specifity of over 90%. ELISA1 had a sensitivity of 99% and ELISA4 a specificity of 99%. Conclusion The sensitivity ELISA1 and the specificit of ELISA4 are comparable to ELISA/WB standard strategy. Application of this alternative testing strategy provides a cost-effective method for determining HIV prevalence in Beijing.