The rapid deployment of emergency tents for airborne disease containment necessitates effective and sustainable approaches. This study introduces an innovative emergency tent prototype, developed within the INITIATE² project by WFP and WHO, that leverages natural ventilation to mitigate airborne transmission risks when humanitarian tents are deployed in response to epidemics. The tent features a two-zone design with a transparent barrier separating the patient area from the healthcare operator zone and exploits a suitable airflow path to reduce cross-contamination. In order to overcome the constraints imposed by the logistic of the on-site measurements, a novel asynchronous single-gas tracer decay methodology combined with a multizone gray box model was developed, enabling both on-site experimental testing of ventilation effectiveness and estimation of airborne pathogen concentrations for infection transmission risk analysis. This approach allowed for the quantification of interzonal exchanges and ventilation rates under various window configurations, simulating different natural ventilation regimes. Multiple ventilation scenarios were evaluated, revealing that partial windows opening (Scenario 2, with Scenario 1 being windows closed) optimized airflow, achieving up to 15 air changes per hour (ACH), a value aligned with CDC and WHO guidelines. Instead, fully open windows (Scenario 3) increased the ACH in the patient area but compromised, to a certain extent, the containment of the pathogens in the healthcare operator zone. Results highlighted, for all the tested scenarios, an unintended air recirculation between the patient and the doctor zones. While the gray box model effectively estimated flow rates across scenarios, it encountered limitations at ACH > 20 due to the photoacoustic equipment’s sampling constraints. The relatively slow acquisition time impacted on the data accuracy during rapid decay phases, where ventilation time constants were on the order of minutes. The design of the transparent barrier reflects a deliberate trade-off between airtightness and operational functionality, with the field methodology enabling an evidence-based assessment of its performance. These findings emphasize the need for refined airflow management and highlight the potential of natural ventilation in emergency healthcare settings. Future research directions include the development of high sampling rate, multigas, and multipoint monitoring tools, as well as enhanced tent designs that improve airtightness of the transparent barrier.