Hemolysis is still the most common reason for rejecting samples, while reobtaining a new sample is an important problem. The aim of this study was to explain the decrease in the activity of Alkaline phosphatase enzyme after hemolysis of blood samples and the possible conversion of zinc and Magnesium ions to inhibitors of alkaline phosphatase activity after they released from red blood cells. Twenty healthy volunteers were enrolled in the study. Four hemolysis levels were constituted according to hemoglobin concentrations (0.02 ,0.27 ,0.75 and 3.34 g/L). Non –hemolysed samples were obtained from each volunteer and considered as control. Hemolysis was achieved by mechanical trauma. Alkaline phosphatase activity and the concentrations of Zinc and Magnesium ions were measured in the hemolysed and non- hemolysed samples. 10 non-hemolysed serum samples (Hb concentration was < 0.02 g/L) were divided in to two groups samples (named as Group A and B ) ALP activity was measured in these samples. In vitro study was carried out through addition of magnesium chloride (68.3 mg/dL) to group A and Zinc chloride (5.1 µg/dL) to group B. ALP activity was measured in the sera of the two groups. The significant decrease (p < 0.001) in ALP activity (13.2 ± 7.2 IU/L) was at moderate and at severe heimolysis ( 5.5 t 2.3 IU/L) as compared with that in non hemolysed samples. In these levels of hemolysis the concentrations of Zn⁺² ions (5.1 ±1.1 µg/dL) and Mg⁺² ions (68.3 ± 8.6 mg/dL) were significantly increased (p < 0.01) compared with their concentrations in non-hemolysed samples (2.75 ± 0.82 ; 17.66 + 2.3µg/ml) respectively. ALP activity was inversely proportional with the increase in the hemoglobin concentrations in the hemolysed samples. A significant decrease (p < 0.005) in the activity of ALP from 93.7 ± 10.2 to 47.4 ±10.7 IU/L was observed after the addition of 68.3.0 mg/dL of magnesium chloride to group. There was no significant decrease (p>0.1) in activity of ALP in the samples of group B to which 5.1 µg/dL of Zinc chloride was added.