Spiroplasma is a genus of wall-less helical bacteria with swimming motility unrelated to conventional types ofbacterial motility machinery, such as flagella and pili. The swimming of Spiroplasma is suggested to be driven by five classes of MreB (MreB1-MreB5), which are members of the actin superfamily. In vitrostudies of Spiroplasma MreBshave recently been conducted to evaluate their activities, such as ATPase, which is essential for the polymerization dynamics among classic actin superfamily proteins. In this chapter, we describe methods of purification and Pi release measurement of Spiroplasma MreBs using column chromatography and absorption spectroscopy with the molecular probe, 2-amino-6-mercapto-7-methylpurine riboside (MESG). Of note, the methods described here are applicable to other proteins that possess NTPase activity.