The doxycycline inducible overexpression system is a highly flexible and widely used tool for both in vitro and in vivo studies. However, during the past decade, a handful of reports have explicitly called for caution when using this system. The raised concerns are based on the notion that doxycycline can impair mitochondrial function of mammalian cells and can alter properties such as cell proliferation. As such, experimental outcomes can be confounded with the side effects of doxycycline and valid interpretation can be seriously threatened. Today, no consensus seems to exist about how these problems should be prevented. Moreover, some of the strategies that have been used to cope with these difficulties can actually introduce additional problems that are related to genomic instability and genetic modification of the cells. Here, we elaborate on the above statements and clarify them by some basic examples taken from our personal wet-lab experience. As such, we provide a nuanced overview of the doxycycline inducible overexpression system, some of its limitations and how to deal with them.