Accelerated healing in wild or captive South American sea lions (Otaria flavescens) is a key tool to help minimize infection and complications associated with open wounds, dental disease, and ocular pathology. Platelet-rich plasma (PRP) is an autogenous source for growth factors based on platelet concentration, which can be obtained by centrifuging whole blood collected in sodium citrate anticoagulant. Currently, there are well-defined PRP concentration protocols for humans and most domestic companion animal species. However, there is no clear centrifugation protocol for obtaining PRP in most marine mammal species. This study aimed to optimize the platelet concentration protocol based on whole blood centrifugation using speeds ranging from 500 to 5,000 rpm and times ranging from 3 to 6 min. Blood was drawn from seven adult South American sea lions, placed into 1-ml sodium citrate tubes, and centrifuged following 12 different centrifugation protocols. PRP was designated as the lower third fraction of the centrifuged plasma. Platelet counts were performed using flow cytometry and statistical analysis was carried out to establish a well-defined protocol for efficient PRP production. Transmission electron microscopy (TEM) analysis was performed to evaluate possible platelet degranulation during the different centrifugation protocols and measure platelet areas. Maximum concentration of platelets in PRP was 4.73-fold higher than the number of platelets in equal volume of whole blood, and significant differences in the concentrations obtained were found between the 12 centrifugation protocols evaluated using different speed and time combinations. The best one-step centrifugation protocol resulted from using 900-rpm speed for 3 min. The highest-fold increase was achieved using a two-step centrifugation protocol, which combined the most efficient one-step centrifugation protocol (900 rpm, 3 min) with a second centrifugation using 2,000-rpm speed for 6 min. TEM analysis confirmed that platelets were complete and maintained integrity after the proposed protocol.