Herbicide active ingredients, formulation type, ambient temperature, and humidity can influence volatility. A method was developed using volatility chambers to compare relative volatility of different synthetic auxin herbicide formulations in controlled environments. 2,4-D or dicamba acid vapors emanating after application were captured in air-sampling tubes at 24, 48, 72, and 96 h after herbicide application. The 2,4-D or dicamba was extracted from sample tubes and quantified using liquid chromatography and tandem mass spectrometry. Volatility from 2,4-D dimethylamine (DMA) was determined to be greater than that of 2,4-D choline in chambers where temperatures were held at 30 or 40 C and relative humidity (RH) was 20% or 50%. Air concentration of 2,4-D DMA was 0.399 µg m−3 at 40 C and 20% RH compared with 0.005 µg m−3 for 2,4-D choline at the same temperature and humidity at 24 h after application. Volatility from 2,4-D DMA and 2,4-D choline increased as temperature increased from 30 to 40 C. However, volatility from 2,4-D choline was lower than observed from 2,4-D DMA. Volatility from 2,4-D choline at 40 C increased from 0.00458 to 0.0263 µg m−3 and from 0.00341 to 0.025 µg m−3 when humidity increased from 20% to 50% at 72 and 96 h after treatment, respectively, whereas, volatility from 2,4-D DMA tended to be higher at 20% RH compared with 50% RH. Air concentration of dicamba diglycolamine was similar at all time points when measured at 40 C and 20% RH. By 96 h after treatment, there was a trend for lower air concentration of dicamba compared with earlier timings. This method using volatility chambers provided good repeatability with low variability across replications, experiments, and herbicides.