Thirty balady rabbits of both sexes (weighing 1.5-2 Kg wt) were used for studding pefloxacin residues. The effect of heat treatment and freezing on the presence of these residues were also studied. The drug was injected intramuscularly (10 mg/Kg body weight) for 5 successive days. Animals were slaughtered at different intervals, samples from shoulder, thigh, back muscle, liver, and kidneys were examined. Parts of samples from animals slaughtered at 12 hr, 24 hr, 48 hr, 72 hr, and 96 hr after treatment, were examined for presence of drug residues and then boiled for 45 minutes and tested while the rest of these samples were frozen and examined weekly for presence of tested drug using HPLC. The result showed that pefloxacin highest concentration level was detected in kidneys at 12 hr from last dose (28.32±2.261 µg/g) and decreased till not detected at 144hr followed by liver (26.32±2.31 µg/g) then shoulder muscle (18.21±1.011 µg/g) and its level showed significant decrease at (p< 0.05) till not detected at 72 hr. concentration level showed in shoulder muscle, thigh and back muscles was (18.21±1.11, 13.5±1.023, 12.6±1.031 µg/g). Regarding the effect of boiling of the drug in kidney sample it is evident that pefloxacin concentration was decreased from 28.32 ±2.261 to 16.516 ±0.421 µg/g). Such decrease was detected in the samples examined at the following hours until disappeared. Meanwhile frozen kidneys sample at -10°C showed significant decrease after 1^st week and decreased till not detected at 5^th week (14.240±0.351, 6.425±0.052, 1.253±0.322 µg/g) followed by liver (4.250±0.33, 2.872±0.251, 1.205±0.158, 0.38±0.241 µg/g).