Background: A laboratory-free test for assessing recovery from pulmonary tuberculosis (TB) would be extremely beneficial in regions of the world where laboratory facilities are lacking. Our hypothesis is that analysis of cough sound recordings may provide such a test. In the current paper, we present validation of a cough analysis tool. Methodology/Principal Findings: Cough data was collected from a cohort of TB patients in Lima, Peru and 25.5 hours of recordings were manually annotated by clinical staff. Analysis software was developed and validated by comparison to manual scoring. Because many patients cough in bursts, coughing was characterized in terms of cough epochs. Our software correctly detects 75.5% of cough episodes with a specificity of 99.6% (comparable to past results using the same definition) and a median false positive rate of 4 false positives/hour, due to the noisy, real-world nature of our dataset. We then manually review detected coughs to eliminate false positives, in effect using the algorithm as a pre-screening tool that reduces reviewing time to roughly 5% of the recording length. This cough analysis approach provides a foundation to support larger-scale studies of coughing rates over time for TB patients undergoing treatment. © 2012 Larson et al.