In vitro penetration has been done on ethanol extract from tuber root of teki grass (Cyperus rotundus L.) with concentration 7% in gel. The penetration test was conducted by two membranes that are whatman paper No.1 with Spangler fluid modification and skin of mice. The test was observed every hour for 3 hours by using Franz diffusion cells. Extract compounds and compounds in penetration liquid were analyzed by GCMS and compared to profile of extract compound. Profile of compounds of both extract and penetration test are same and showed contain some volatile compounds such caryophyllene oxide, Sphatulenol, Alloaromadendrene, Iso-velerenal, 1-Limonene, Globulol, Beta-Guaiene, and Beta-Citronellol. Based on this assay can be concluded that ethanol extract of Cyperus rotundus in gel formula can be penetrated through two membranes that are Spangler and skin of mice.