Amikacin is a semisynthetic bactericidal aminoglycoside derived from kanamycin A with a broad spectrum of activity. The aim of this work is to investigate the withdrawal of amikacin residues in the tissues of rabbits and the effect of boiling and freezing on amikacin residues using high performance liquid chromatography (HPLC). About 15 rabbits were injected with an intramuscular dose of amikacin (15 mg/kg BW) for 7 successive days. Samples were obtained from kidneys, liver and breast muscle on the 1st, 3rd, 5th, 7th, and 10th day after the last dose. Sample extraction was carried out using solid phase extraction and derivatization process. Reversed HPLC analysis included fluorescence detection at an excitation wave-length 360 nm and emission 435 nm and mobile phase mixture (69: 31 v/v) of methanol, water with a flow rate 1.5 min. Our results revealed that residues of amikacin in the fresh kidneys, liver and breast muscle were 14.2± 0.57 µg/g, 9.02± 0.45µg/g, and 8.23± 0.18µg/g, respectively in the first day post-treatment. The residues' level was declined to 0.19 ± 0.02 in kidney and not detected in liver and breast muscle on the 10th day post treatment. Residues level in rabbitsʹ kidneys, liver and breast muscle was recorded as a reduction % of 26.3%, 32.35%, and 44.13% after boiling treatment and 11.74%, 20.18%, and 22.27% after freezing, respectively compared to the residues in fresh tissues in the first day post treatment. The obtained results concluded that the withdrawal time of amikacin residues from the tissues of rabbits is 10 days.