Natural Killer T (NKT) cells, a specialized subset of T cells, play a pivotal role in immune surveillance. They recognize lipid antigens presented by the MHC class I-like molecule CD1d, bridging innate and adaptive immunity. NKT cells exhibit both pro-inflammatory and immunoregulatory properties, impacting various pathological states, including infections, autoimmune diseases, and cancer. The NKT hybridoma cell stimulation assay is a powerful tool for detecting and characterizing NKT cells, as well as screening natural and synthetic lipid antigens, potentially leading to novel therapeutic interventions. Here, we describe two complementary methods (cell-free and cell-based) to stimulate NKT hybridoma cells to secrete cytokine, which serves as an indirect measure of NKT cell activation. These assays provide insight into the intricate interplay between NKT cells, lipids, and other immune cell subsets, including antigen-presenting cells, and their impact on immune regulation and disease progression.