Sampling methods of methane concentration (CH4) of gas produced from in vitro fermentation (IVGP) were evaluated to assess their determination efficacy. The original protocol recommends directly placing fermented bottles on ice (0C) for 30 minutes to stop fermentation (D). An alternate protocol recommends placing the fermented bottles into the refrigerator (46C) to slow fermentation (S). This experiment evaluated the previous methods against direct sampling of the gas after 48 h of fermentation at 39C (I). Rumen inoculum was pulled from four rumen cannulated steers and filtered through fiberglass wool. Ground alfalfa was used as the fermentable substrate and total gas production was recorded for 48 h of fermentation. After fermentation, each bottle followed a randomly assigned protocol. The pressure and volume of gas in the bottle were recorded, 12 mL of gas from the headspace was placed into an evacuated exetainer for (CH4) sampling via gas chromatography, and the temperature of the fermented fluid was recorded. Eight bottles from D and eight bottles from S were randomly selected to follow the exetainer protocol, while the remaining bottles had (CH4) directly measured from their headspace. Statistical analysis was completed using a random coefficients model. Methane concentration was higher for I than D (P = 0.0286) and S (P = 0.0070). There was no difference in (CH4) between D and S (P = 0.5286). There was no difference in (CH4) in D exetainers and bottles (P = 0.5744), but there was a difference in (CH4) in S exetainers and bottles (P = 0.0229). Pressure, volume, and temperature were different among all protocols (P 0.0311). Based upon the data, protocol I provides the best estimate of (CH4). Further research is required to understand the discrepancy of (CH4) among the protocols relative to temperature, pressure, and volume.