Chlorella sp. is a prospective steroids source which is potentially applied in pharmaceutical field. The aim of this research was to isolate and determine the toxicity and antioxidant activity of steroids compounds in petroleum ether (PE) fraction. Chlorella sp. was cultivated in 4% of sprout extract medium and extracted by maceration using methanol solvent. The methanol extract was partitioned with petroleum ether and the fraction was identified by LC-MS to know the type of steroids compound. The PE fraction contained steroids was separated with preparative thin layer chromatography (Preparative TLC) and column chromatography. The toxicity level and antioxidant assay of steroids isolates was determined by BSLT and DPPH method, respectively. According to phytochemical test and LC-APCI-MS identification, the PE fraction was positive for steroids compounds such as β-sitosterol, stigmasterol, campesterol and erythrodiol. Steroid isolates from TLC and column chromatography separation has toxicity and antioxidant properties. Based on toxicity test, LC50 value of steroid isolate by TLC separation was 19.69 ppm, whereas, by column chromatography separation, LC50 value of A10 and A12 isolate was 26.67 and 49.04 ppm, respectively. Antioxidant activity (EC50 value) of steroid isolate by TLC separation was 73.82 ppm. Percentage of antioxidant activity of A2, A8, A10 and A12 isolates (by column chromatography separation) at 50 ppm were 2.60, 4.09, 4.52 and 3.29 %.

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