The use of X-rays for non-invasive imaging has a long history, which has resulted in several well-established methods in preclinical as well as clinical applications, such as tomographic imaging or computed tomography. While projection radiography provides anatomical information, X-ray fluorescence analysis allows quantitative mapping of different elements in samples of interest. Typical applications so far comprise the identification and quantification of different elements and are mostly located in material sciences, archeology and environmental sciences, whereas the use of the technique in life sciences has been strongly limited by intrinsic spectral background issues arising in larger objects, so far. This background arises from multiple Compton-scattering events in the objects of interest and strongly limits the achievable minimum detectable marker concentrations. Here, we review the history and report on the recent promising developments of X-ray fluorescence imaging (XFI) in preclinical applications, and provide an outlook on the clinical translation of the technique, which can be realized by reducing the above-mentioned intrinsic background with dedicated algorithms and by novel X-ray sources.