Bartonella bacilliformis causes bartonellosis, a potentially life-threatening emerging infectious disease seen in the Andes Mountains of South America. There are no generally accepted serologic tests to confirm the disease. We developed an indirect fluorescence antibody (IFA) test for the detection of antibodies to B. bacilliformis and then tested its performance as an aid in the diagnosis of acute bartonellosis. The IFA is 82% sensitive in detecting B. bacilliformis antibodies in acute-phase blood samples of laboratory-confirmed bartonellosis patients. When used to examine convalescent-phase sera, the IFA is positive in 93% of bartonellosis cases. The positive predictive value of the test is 89% in an area of Peru where B. bacilliformis is endemic and where the point prevalence of infection is 45%.