Mitochondria are not only the "power-house" of the cell, but are also involved in a multitude of processes that include calcium storage, cell-cycle and cell-death. Traditional means to investigate mitochondrial importance in a given cellular process have centred upon depletion of mitochondrial DNA (mtDNA) through chemical or genetic means. While these methods severely disrupt mitochondrial electron transport chain, mtDNA-depleted cells still maintain mitochondria and many mitochondrial functions. Here, we describe a straightforward protocol to generate mammalian cell populations with low to non-detectable levels of mitochondria. Ectopic expression of the ubiquitin E3 ligase Parkin, combined with short-term mitochondrial uncoupler treatment, engages widespread mitophagy, and effectively eliminates mitochondria. In this protocol, we explain how to generate mitochondria-depleted cells and a variety of methods to confirm mitochondrial clearance. Furthermore, we describe culture conditions to maintain mitochondrial-depleted cells with minimal loss of viability for longitudinal studies. This method should prove useful to investigate the importance of mitochondria in a variety of biological processes.