Platelet rich plasma (PRP) is popularly used in the horse industry to enhance regeneration of tissue injury that has limitation of blood supply. This study aimed to compare the methods for platelet rich plasma preparation since they has not been established yet. Blood was collected from six horses and platelets were concentrated by three different methods (2-step centrifugation, separated centrifugation and separated centrifugation using histopaque). Concentrated blood was analyzed using Advia hematology systems. In the result, separated centrifugation with histopaque showed the significantly lower number of red blood cells than other groups. The 2-step centrifugation showed the significantly higher number of white blood cells than other groups, while it contained the highest concentration of red blood cells among three groups. In the 2-step centrifugation, separated centrifugation and separated centrifugation with histopaque, platelets were concentrated 4.5, 5.3 and 5.6 times, respectively. And no significant difference of the platelet concentration between the three groups was found. This study demonstrated that separated centrifugation using histopaque was the best method for platelet rich plasma preparation because of the proper amount of platelets and the separation of red blood cells from platelet rich plasma.