The objective of this study was to evaluate the effect of the level of cashew nut shell oil (CNSO) on methane production in an in vitro incubation. The experimental design was a 3*4 factorial with 3 replications. The first factor was feed source (Para grass, Sesbania grandiflora and Operculina turpethum) and the second factor was supplemental levels of CNSO (0, 1, 2, 3 and 4 %, DM basis). The substrates (2.5g) were added 200ml of buffer solution and 50ml of buffalo rumen fluid taken immediately after the animal was killed in the slaughter-house. The incubation was for 96h with measurements of gas and methane production at 6, 12, 24, 48, 72 and 96h. Unfermented solids at 96 h were determined by filtering through two layers of cloth and drying at 105°C for 24 h and ashing for 5 hrs to measure the DMD and OMD. The results of this paper showed that the total gas production, CH4 production and concentration after 96h incubation were significantly different (Pin vitro incubation with the higher reduction at 4% of supplemental levels.