Cell culture, particularly two-dimensional (2D) cell culture, has been widely used for in vitro MG and neuromuscular junction (NMJ) physiology studies, as they are user-friendly, widely accessible, and inexpensive. These conventional models have been used since the 1970s (Peterson & Crain, 1972) to understand the molecular mechanisms underlying NMJ formation and maintenance and the pathophysiology of neuromuscular disorders such as MG. While these models have led to significant insights, it must be noted that conventional 2D models lack structural context and are, therefore, unsuitable for examining the mechanical aspects of MG pathophysiology, such as muscle weakness. In addition, muscle cells in culture will start spontaneous contractions after five days (Peterson & Crain, 1972), often limiting the longevity of such cultures without additional treatments (Manabe et al., 2012, Park et al., 2008). Therefore, newer models have arisen, e.g. 3D systems and NMJ organoids, which provide a microenvironment that mimics the native extracellular matrix (ECM) and allows cells to interact with each other in a manner that more closely represents the in vivo NMJ, allowing for functional tests and for more long-lived cultures (Langer & Vacanti, 1993). In these models, the myasthenic phenotype can be recapitulated by introducing patient serum or purified antibodies from patients with acetylcholine receptor antibodies (AChR+ MG), muscle-specific tyrosine kinase antibodies (MuSK+ MG), or seronegative MG (SNMG) patients. Such MG disease modeling has been conducted in different cell-based settings, providing valuable insights into the molecular pathogenic mechanism. In this chapter, we give an overview of in vitro models used for modeling the autoimmune attack at the NMJ in MG and summarize the readouts that can be used to analyze these models for pre-clinical MG studies (Fig. 1).