Background: Hydroalcoholic extract from aerial part of Pycnocycla spinosa have shown to possess antispasmodic and anti-diarrhoeal activities. Anti-diarrhoeal effect of the extract, both quantitatively and qualitatively, are very close to loperamide. The aim of this research was identification of active fraction separated from extract of Pycnocycla spinosa via applying bioassay method on contraction of rat ileum.Methods: From aerial part of Pycnocycla spinosa methanolic, extract was prepared and then medium polar fractions were separated. The antispasmodic activities of separated fractions were assessed on rat isolated ileum contraction induced by KCl and compared with total extract. After that, the most active fraction was identified for further comparison with control group. Contractions were measured as maximum recorded amplitude and express as percent of initial contraction. Full concentration response curve was plotted for each group of experiment and the IC50 value (inhibitory concentration of extract causing 50% of maximum response) was calculated.Findings: From nine fractions isolated from Pycnocycla spinosa extract, the third fraction identified as the most active fraction which contained the active substance(s). Five fractions had a medium activity and two fractions were ineffective.Conclusion: This research demonstrated that Pycnocycla spinosa possessed a number of active substances with different potencies. The most potent substance was in the third fraction and therefore, purification and identification of this substance is recommended.