The diagnostic workup for myasthenia gravis extends beyond the objective evaluation of skeletal muscle fatigue during neurological examinations. It incorporates antibody testing, electrophysiological studies to confirm neuromuscular transmission impairment, and chest imaging to detect thymoma. Positive clinical response to acetylcholinesterase inhibitors may also support diagnosis. Key clinical assessments focus on symptoms of skeletal muscle fatigability, particularly in ocular, bulbar, and limb-girdle muscles. While the ice-pack test is commonly used to assess ptosis, the global availability of acetylcholinesterase inhibitors for testing remains limited. Radioimmunoassay is the most sensitive diagnostic method for MG-specific antibodies, such as AChR and MuSK, followed by cell-based assays that utilize clustered receptors. Enzyme-linked immunosorbent assay (ELISA) is another option, though with reduced specificity. Electrophysiological evaluation begins with repetitive nerve stimulation (RNS) to detect postsynaptic transmission failure, with single-fiber electromyography (SFEMG) employed in cases where RNS results are inconclusive.

All patients with MG, regardless of subtype, should undergo chest imaging (CT or MRI) to screen for thymoma. Differential diagnoses to consider include congenital myasthenic syndromes, cranial nerve disorders such as Horner syndrome or third nerve palsy, autoimmune demyelinating polyneuropathy, mitochondrial myopathy, and motor neuron disorders.