Abstract: In the biological sciences, model organisms have been used for many decades and have enabled the gathering of a large proportion of our present-day knowledge of basic biological processes and their derailments in disease. While in many of these studies using model organisms, the focus has primarily been on genetic and genomic approaches, it is important that methods become available to extend this to the relevant protein level. Mass spectrometry-based proteomics is increasingly becoming the standard to comprehensively analyze proteomes. An important transition has been made recently by moving from charting static proteomes to monitoring their dynamics by simultaneously quantifying multiple proteins obtained from differently treated samples. Especially the labeling with stable isotopes has proved an effective means to accurately determine differential expression levels of proteins. Among these, metabolic incorporation of stable isotopes in vivo in whole organisms is one of the favored strategies. In this perspective, we will focus on methodologies to stable isotope-label a variety of model organisms in vivo, ranging from relatively simple organisms as bacteria and yeast, via C. elegans, Drosophila and Arabidopsis up to mammals such as rats and mice. We also summarize how this has opened up ways to investigate biological processes at the protein level in health and disease, revealing conservation and variation across the evolutionary tree of life.