Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. It is involved in numerous important brain functions such as learning, memory and cognition, as well as the development and plasticity of the central nervous system. In order to ensure efficient signal transmission, glutamate is highly compartmentalized. Prolonged elevated extracellular levels of glutamate have been shown to be excitotoxic with the result of neuronal cell death ultimately. Furthermore, alterations in glutamate levels have been shown to be linked to several neurodegenerative disorders such as Alzheimer's, Parkinson's and Huntington's diseases, as well as ischemic stroke and amyotrophic lateral sclerosis. Accurate measurement of glutamate levels in vitro and in vivo for a better understanding of the physiological and pathological role of glutamate in neurotransmission has remained challenging, and different techniques have been developed to this end. This review presents and discusses these techniques, especially the recent progress in the field of glutamate biosensors, as well as the great potential of nanotechnology in glutamate sensing. Microdialysis coupled to analytical detection techniques is currently the most common method for in vivo glutamate sampling. However, the recent development and improvement of enzyme-based amperometric glutamate biosensors makes them a promising alternative to microdialysis for in vivo applications, as well as valuable devices for in vitro applications in basic neurobiological research. Another interesting group of biosensors for glutamate are fluorescence-based glutamate biosensors, which have unsurpassed spatio-temporal resolution and are therefore important tools for investigating glutamate dynamics during signaling. Adding to this list are biosensors based on nano-materials such as nanoparticles, nanotubes and nanowires, which have great potential for sensing applications due to their large surface-to-volume ratio and novel physical properties.