Platelets are metabolically active, anucleated and small circulating cells mainly responsible for the prevention of bleeding and maintenance of hemostasis. Previous studies showed that platelets mitochondrial content, function, and energy supply change during several diseases such as HIV/AIDS, COVID-19, pulmonary arterial hypertension, and in preeclampsia during pregnancy. These changes in platelets contributed to the severity of diseases and mortality. In our previous studies, we have shown that the seahorse-based cellular stress assay (CSA) parameters are crucial to the understanding of the mitochondrial performance in peripheral blood mononuclear cells (PBMCS). Moreover, the results of CSA parameters were significantly influenced by the PBMC preparation methods. In this study, we assessed the correlation of CSA parameters and intracellular ATP content in platelets and evaluated the effects of platelet preparation methods on the results of CSA parameters and intracellular ATP content. We compared the results of CSA parameters and intracellular ATP content in platelets isolated by density centrifugation with Optiprep and simple centrifugation of blood samples without Optiprep. Platelets isolated by centrifugation with Optiprep showed a higher spare capacity, basal respiration, and maximal respiration than those isolated without Optiprep. There was a clear correlation between basal respiration and maximal respiration, and the whole-ATP content in both isolation methods. Moreover, a positive correlation was observed between the relative spare capacity and whole-cell ATP content. In conclusion, the results of seahorse-based CSA parameters and intracellular ATP content in platelets are markedly influenced by the platelet isolation methods employed. The results of basal respiration and maximal respiration are hallmarks of cellular activity in platelets, and whole-cell ATP content is a potential hint for basic platelet viability. We recommend further studies to evaluate the role of CSA parameters and intracellular ATP content in platelets as biomarkers for the diagnosis and prediction of disease states.