A simple and expeditious analytical method for determination of zinc in human insulin isophane suspension by flame atomic absorption spectrophotometer (FAAS) was validated. The method was carried out on atomic absorption spectrometer with 0.4nm bandwidth, 1.0 filter factor on deuterium (D2) background correction. The integration time was set at 3.0 second with 5.0mA lamp current. The parameters of method validation showed adequate linearity, efficiency and relative standard deviation values were between 0.64%-1.69% (n=7), 1.31%-1.58% (n=10) for repeatability and intermediate precision respectively. The limit of detection 0.0032µg/mL, 0.0173µg/mL, 0.0231µg/mL and limit of quantitation 0.0107µg/mL, 0.0578µg/mL, 0.0694µg/mL based on signal to noise (SN), calibration curve method (CCM) and fortification of blank (FB) were obtained respectively. The percentages of recovery for low, medium and high spiked concentration levels of zinc in human insulin were 99.38±0.04 to 100.3±0.03, 98.45±0.38 to 100.3±0.07 and 99.42±0.03 to 99.42±0.08 respectively. With the use of this method, five samples from each vial of human insulin isophane suspension were analyzed and the zinc content was determined. The zinc content were 22.1±0.025µg/mL and 24.3±0.028 µg/mL which compliance the British Pharmacopoeia standard.