Saccharomyces cerevisiae is one of the most popular expression systems for eukaryotic membrane proteins. Here, we describe protocols for the expression and purification of mitochondrial membrane proteins developed in our laboratory during the last 15 years. To optimize their expression in a functional form, different promoter systems as well as codon-optimization and complementation strategies were established. Purification approaches were developed which remove the membrane protein from the affinity column by specific proteolytic cleavage rather than by elution. This strategy has several important advantages, most notably improving the purity of the sample, as contaminants stay bound to the column, thus eliminating the need for a secondary purification step, such as size exclusion chromatography. This strategy also avoids dilution of the sample, which would occur as a consequence of elution, precluding the need for concentration steps, and thus preventing detergent concentration.