Purification of skeletal muscle myoblasts from other cells such as fibroblasts is essential inbasic research and cell-based regenerative medicine. In this report a simple non-serologicalmethod is proposed to purify desmin-positive myoblasts using Brefeldin A. Ordinary passaged human skeletal muscle-derived cells were cultured for 3 days with fiveexocytic pathway inhibitors (Brefeldin A, Monensin, AACOCF3, Exo-1 and Golgicide A). The myoblasts identified by desmin exhibited relatively superior growth compared to the other cells, both in 7 - 15 ng/mL of Brefeldin A and in 850 – 2500 ng/mL of Golgicide A. In the case of 10 ng/mL Brefeldin A, the cell numbers increased 6.7-fold in desmin-positive cells and 2.4-fold in desmin-negative cells while they increased 11.7-fold and 15.6-fold in absence of Brefeldin A, respectively. In addition, 7-day cultures in 10 ng/mL Brefeldin A of four independent cell strains suggested that moderate starting purities resulted in high purities over 90%, while extremely low starting purities (< 1%) did not support stable purifications. Lastly, a strain purified through a 7-day culture with 10 ng/mL Brefeldin A was subjected to myotube induction culture. Fluorescent imaging using a confocal microscope revealed their ability to form multinucleated myotubes and myofibers with sarcomeric structures. In conclusion, the purification of desmin-positive cells by culturing human skeletal musclederived cells with Brefeldin A provides a simple method to obtain purified myoblasts for use in basic research and cell-based regenerative medicine.