A new method for rapid sampling and qualitative characterization of the headspace gases of closed in vitro cultures using solid phase microextraction (SPME) was evaluated for ruminal fluid and ruminal fluid with feed containing a feed additive. Gas sample collection was achieved by exposing a DVB/Carboxen/PDMS 50/30 .m SPME fiber to the headspace of cultures for 1 min every hour. This was followed by immediate analysis on a GC–MS and then reuse of the SPME fiber. Acetic, propionic, isobutyric, butyric, isovaleric, valeric, and hexanoic acids and toluene, dimethyl disulfide, and pentadecane were identified in the headspace. SPME technology facilitated rapid sampling and immediate analysis with a GC–MS to identify specific end products of microbial digestion. A SPME–based approach could serve as a new method for the characterization of ruminal fermentation end products and testing effects of feed additives on the formation of end products.