Low-salinity stress can severely affect the fitness of marine organisms. As desalination has been predicted for many coastal areas with ongoing climate change, it is crucial to gain more insight in mechanisms that constrain salinity acclimation ability. Low-salinity induced depletion of the organic osmolyte pool has been suggested to set a critical boundary in osmoconforming marine invertebrates. Whether inorganic ions also play a persistent role during low-salinity acclimation processes is currently inconclusive.We investigated the salinity tolerance of six marine invertebrate species following a four-week acclimation period around their low-salinity tolerance threshold. The species investigated were Asterias rubens, Mytilus edulis, Littorina littorea, Diadumene lineata, Strongylocentrotus droebachiensis and Psammechinus milliaris. To obtain complete osmolyte budgets of seawater, body fluids and tissues we quantified total osmolality (via osmometer), organic osmolytes (methylamine and free amino acids) via 1H-NMR spectroscopy and inorganic osmolytes (anions and cations) via flame photometry and a novel protocol using ion-chromatography. We further determined the fitness proxies survival, growth and tissue water content.Our data show the importance of the organic and inorganic osmolyte pool during low-salinity acclimation. It also shows the importance of specific compounds in some species.This data can be used in future osmolyte and salinity tolerance research. This type of data is essential to establish reliable physiological limits of species in order to estimate consequences of future salinity changes with ongoing climate change. It can be used to assess the salinity tolerance capacity and to obtain a better understanding of the basic mechanisms that are utilized in a wide range of species. The established cellular inorganic and organic osmolyte profiles can build a foundation for applied cellular physiological research, for example for designing suitable buffers for in vitro assays as these buffers need to incorporate complex organic and inorganic osmolyte changes. Knowledge about cellular and whole-organism biochemistry and physiology is absolutely crucial for characterizing the functions of genes that are under selection by climate change stressors. A quantitative knowledge of cellular osmolyte systems is key to understand the evolution of euryhalinity and to characterize targets of selection during rapid adaptation to ongoing desalination.