Mitochondria are hot! First of all, literally. Recent results showed that locally in the functional human mitochondria, the temperature rises close to 50°C. The research was based on the thermosensitive fluorescent dye that targets mitochondria. Figuratively, mitochondria are even hotter, in terms of undergoing research exploring their role in energy production, cell signalling, programmed cell death and biosynthesis. Fluorescent probes and dyes are not restricted only to imaging of these fascinating organelles; they are used to monitor the mitochondrial membrane potential, pH and the redox status. The membrane potential is essential in the process of ATP production as it constitutes more than 80% of electrochemical proton motive force used in this process. Thus, observing mitochondrial membrane potential is crucial in most of the mitochondrial research. This imaging should usually be performed with minimal invasiveness and damage to the mitochondria and mitochondrial function. There are only a few fluorescent probes for mitochondrial membrane potential monitoring currently in use as valuable indicators of cells’ functional status. Those probes show varying degrees of interference with cell or mitochondrial metabolism and photo-induced damage. In this chapter, the characteristics, advantages and disadvantages of such probes will be discussed. The mechanisms of uptake of these fluorescent probes will be explained.