Indoor air quality (IAQ) data of 220 classrooms in the Midwest region of the United States were measured during 2015-2017. To calculate ventilation rates from the collected CO2 data, there are three main methods derived from the mass balance equation: (1) steady-state; (2) decay rate; and (3) build-up methods. Since the uncertainty of the individual measurement parameter would affect the accuracy or the reliability of the calculated ventilation rate, an uncertainty analysis was performed for all three methods. R programming was used to calculate the uncertainty of the calculated ventilation rates to the volume of the classroom, indoor and outdoor CO2 levels, and estimated student CO2 emission rates. The study shows that the steady-state method has the minimum uncertainty when calculating ventilation rates while decay and build-up methods have the lowest and highest values for ventilation rates, respectively.