Functional genomic approaches, such as proteomics, greatly enhance the value of genome sequences by providing a global level assessment of which genes are expressed, when genes are expressed and at what cellular levels gene products are synthesized. With over 1000 complete genome sequences of different microorganisms available, and DNA sequencing for environmental samples (metagenomes) producing vast amounts of gene sequence data, there is a real opportunity and a clear need to generate associated functional genomic data to learn about the source microorganisms. In contrast to the technological advances that have led to the accelerated rate and ease at which DNA sequence data can be generated, mass spectrometry based proteomics remains a technically sophisticated and exacting science. In recognition of the need to make proteomics more accessible to a growing number of environmental microbiologists so that the ‘functional genomics gap’ may be bridged, this review strives to demystify proteomic technologies and describe ways in which they have been applied, and more importantly, can be applied to study the physiology and ecology of extremophiles.