Non-specific Gd-based contrast agents (GBCAs) have been used in clinical magnetic resonance imaging (MRI) for more than 35 years. However, the interaction of such contrast agents with tissue components is not yet fully understood. Many diseases, including inflammation, fibrosis, and tumour invasion, are associated with characteristic changes of the extracellular matrix (ECM). The ECM is a three-dimensional network of macromolecules providing structural and biochemical support of the surrounding cells in all mammalian tissues. It is composed of structural proteins (e.g., collagen, elastin) and proteoglycans, which consist of glycosaminoglycans (GAGs) covalently bound to a protein core. GAGs are long, linear polysaccharides composed of repeating disaccharide units that differ in molecular mass, disaccharide structure and degree of sulfation. GAGs are also characterized by their ability to form complexes with cations such as lanthanides. Thus, GAGs could be a potential binding partner for GBCAs as a whole or for dechelated Gd. Laser ablation inductively coupled plasma time-of-flight mass spectrometry (LA/ICP-ToF-MS) was used to investigate the uptake and distribution of ionic Gd and various linear and macrocyclic GBCAs in spheroids mimicking biological tissue and exhibiting different ECM expressions. In addition to Gd, other relevant elements such as Cu, Fe, P and Zn were also monitored. Gelatine doped with multi-element solution was used for matrix-matched quantification. Spheroids from Chinese hamster ovary (CHO) cells and GAG-depleted CRL-2242 cells were incubated with gadolinium chloride and various GBCAs. Although all spheroids were exposed to identical Gd concentrations, differences were observed in the spatial distribution and the amount of Gd taken up. After incubation with linear and macrocyclic GBCAs, Gd is detected in the interior of both types of spheroids. Furthermore, differences in the Gd amount were found depending on the GBCA used. In contrast, incubation with gadolinium chloride leads to an enrichment in the outer regions of the spheroids as well as to much higher Gd contents compared to incubation with GBCAs. LA/ICP-ToF-MS can make an important contribution to better understand the relationship between the affinity of GBCAs and ECM components such as GAGs. However, to elucidate such complex interactions, further studies are needed, also with other (bio-)analytical techniques.