The appropriate use of clinically accurate diagnostic tests is essential for the detection of pertussis, a poorly controlled vaccine-preventable disease. The purpose of this study was to estimate the sensitivity and specificity of different diagnostic criteria including culture, multi-target polymerase chain reaction (PCR), anti-pertussis toxin IgG (IgG-PT) serology, and the use of a clinical case definition.\u00a0 An additional objective was to describe the optimal timing of specimen collection for the various tests.\u00a0Clinical specimens were collected from patients with cough illness at seven locations across the United States between 2007 and 2011. Nasopharyngeal and blood specimens were collected from each patient during the enrollment visit.\u00a0 Patients who had been coughing for \u2264 2 weeks were asked to return in 2-4 weeks for collection of a second, convalescent blood specimen.\u00a0 Sensitivity and specificity of each diagnostic test were estimated using three methods\u2014pertussis culture as the \u201cgold standard,\u201d composite reference standard analysis (CRS), and latent class analysis (LCA).\u00a0Overall, 868 patients were enrolled and 13.6% were B. pertussis positive by at least one diagnostic test. \u00a0In a sample of 545 participants with non-missing data on all four diagnostic criteria, culture was 64.0% sensitive, PCR was 90.6% sensitive, and both were 100% specific by LCA.\u00a0 CRS and LCA methods increased the sensitivity estimates for convalescent serology and the clinical case definition over the culture-based estimates.\u00a0 Culture and PCR were most sensitive when performed during the first two weeks of cough; serology was optimally sensitive after the second week of cough.Timing of specimen collection in relation to onset of illness should be considered when ordering diagnostic tests for pertussis. Consideration should be given to including IgG-PT serology as a confirmatory test in the Council of State and Territorial Epidemiologists (CSTE) case definition for pertussis.