Isolation of leaf mesophyll protoplasts from tobacco (Nicotiana tabacum) is facilitated using a specially designed digestion chamber. The chamber's airtight seals and various ports reduce the complexity, time and contamination risks which may be associated with standard isolations. The polished glass viewing window allows continuous monitoring of protoplast release, thus facilitating more precise determinations of the optimal digestion time. In concert with a simplified centrifugation step, the protoplast isolation procedure is greatly standardized.