Aflatoxins are one of the major factors that affect the quality and safety of feeds that are transferred into livestock species and then to humans via animal sources of food (meat, egg and milk). The objective of this study was to detect and quantify the level of aflatoxins (B1, B2, G1, G2 and total aflatoxin) in spent grains produced in selected brewery companies in Addis Ababa and , animal farms from Adis ababa and sheger. A total of 24 samples consisting of 18 samples collected from selected animal farms of Addis Ababa Shagar city and 6 samples from brewery companies of Addis Ababa and sheger. Samples were analyzed using High Performance Liquid Chromatography after cleaned up with immuno affinity columns. In the spent grain sample analyzed for main effect for place the aflatoxin level B1 obtained from brewery(0.010μg/kg) greater than animal farms(0.0607 μg/kg ); the aflatoxin level B2 obtained from brewery(0.107 μg/kg) higher than animal farm(0.101 μg/kg) ;the aflatoxin G1 obtained from brewery (0.09 μg/kg) lower than animal farms(0.117 μg/kg); the aflatoxin G2 obtained from brewery(0.14 μg/kg) higher than animal farms(0.2 μg/kg) and total aflatoxin obtained from brewery(0.384μg/kg) lower than animal farms(0.433 μg/kg). The analysis of sample on time effect showed that aflatoxin level B1 on first day, after 15 days and 30 days were 0, 05 μg/kg, 0.13 μg/kg and 0.075 μg/kg respectively. Aflatoxin level B2 on first day, 15 days and 30 days were 0.101μg/kg, 0.15μg/kg and 0.06μg/kg respectively. Aflatoxin level G1 on first day, 15 days and 30 days were 0.05 μg/kg, 0.15 μg/kg and 0.11 μg/kg respectively. Aflatoxin level G2 on first day, 15 days and 30 days were 0.2 μg/kg, 0.2 μg/kg, and 0.11 μg/kg respectively and total aflatoxin level on first day,15 days and 30 days were 0.3 μg/kg,0.6 μg/kg and 0.33 μg/kg respectively. 100 % spent grain contained aflatoxin were lower than maximum limit set by FDA and ESA. In conclusion spent grain analyzed from both brewery companies and animal farms were free from aflatoxin contamination. So they should maintain good handling of spent grain. Key words: Aflatoxin, Spent grain, Storage time, Storage place, HPLC