The diagnosis of amyotrophic lateral sclerosis (ALS) remains a clinical diagnosis. It is based on the combination of both upper and lower motor neuron signs in the neurologic examination. With several new therapeutic agents on the horizon, effective and objective disease markers for diagnosis and surrogate outcome measures in clinical trials are crucial. Whereas the presence of lower motor neuron signs on neurologic examination can be ascertained by electromyography, there is no widely accepted marker for upper motor neuron involvement. Neuroimaging changes of the corticospinal tract in ALS patients have been studied using magnetic resonance (MR) imaging, but appear to lack sensitivity and specificity. MR spectroscopy, a technique that allows one to evaluate biochemical tissue composition in vivo, has been widely used to establish the progressive decrease in N-acetylaspartate, a marker of neuronal integrity, in the course of ALS. More recently, diffusion tensor imaging, a newer MR technique, has demonstrated changes in diffusivity along the corticospinal tract in ALS patients. Metabolic aspects in the brains of ALS patients have been evaluated using positron emission tomography. Transcranial magnetic stimulation is a more established technique that evaluates the neurophysiologic integrity of upper motor neurons in ALS. This article reviews the progress that has been made over the past two decades towards establishing valid diagnostic and natural history markers of upper motor neuron involvement in ALS.