Energy metabolism, particularly mitochondrial function, has emerged as a key regulator of the immune response and plays a significant role in the pathophysiology of inflammatory diseases. Studying mitochondrial parameters is therefore crucial in understanding these pathologies. This protocol outlines the evaluation of mitochondrial respiration parameters using a Seahorse Analyzer. With the growing use of innovative models that better mimic human physiology, such as cell cultures in inserts, this chapter provides a detailed guide to Seahorse analysis for this specific type of culture.