Abstract: Sida rhombifolia L., or Sidaguri in Indonesian, is a traditional medicinal plant with potential as a remedy against gout. The earlier research reported that flavonoids crude extract from this plant could in vitro inhibit the activity of Xanthine Oxidase (XO) (xanthine: oxygen oxidoreductase EC 1.2.3.2) up to 55% and could be antigout. It is a need to investigate type of inhibition kinetic of Sida rhombifolia L.’s extract and the effects of fractionation on the inhibitory effect by in vitro method. The results showed that the flavonoids crude extract yielded approximately 12% with LC50 of 501 mg L-1 and its inhibitory effect from 48 to 71% (100-800 mg L-1). The kinetic study resulted that the type of flavonoids crude extract inhibition was a competitive inhibition with inhibitor affinity (α) of 2.32 and p<0.01. The fractionation yielded 11 fractions and the fraction 4 has highest activity, i.e., 79%. The fractions did not increase the inhibition effect significantly. GC-MS analysis of fraction 4 showed that there were 39 organic compounds and flavonoid fragments with retention times of 4.14, 6.53 and 6.74 that have resemble fragmentation of benzoic acid compound. By phytochemical assay, fraction 4 contain flavonoid.