Rho family GTPases are key regulators of cytoskeletal dynamics and control all aspects of cellular morphogenesis, ranging from migration to adhesion. They function as molecular switches that cycle between an inactive GDP-bound state and an active GTP-bound state, in which they can engage with numerous downstream effectors. Rho signalling requires tight spatiotemporal control which is mediated by in total 145 Rho guanine nucleotide exchange factors (RhoGEFs) and Rho GTPase activating proteins (RhoGAPs) in humans. These proteins encode a broad spectrum of targeting and protein interaction domains and thereby contribute specificity to Rho signalling. During development, wound healing or metastasis, cells must change their positions within organs or the entire body. This is achieved by guided cell migration, where attractive or repulsive signals in the extracellular environment direct cells towards distinct positions. A well-studied class of guidance molecules is the SLIT ligand family that upon binding to their ROBO recep- tors directs repulsive migration in diverse tissues. The underlying processes are orchestrated by Rho GTPases, however, the downstream RhoGEFs and RhoGAPs that link the receptors to the GTPases are only poorly characterized and also have not been fully identified. Moreover, for the structurally distinct endothelial-specific ROBO4 receptor so far no regulator has been found at all. In Drosophila melanogaster the RhoGAP Vilse/Crossgap, which is the homologue of the novel human RhoGAP ARHGAP39, has been linked to Robo signalling, as its depletion causes misrouting of tracheal ganglionic branches and axons in the central nervous system (CNS). Furthermore in a previous mass spectrometry screen for RhoGEF/GAP interactors (Müller et al., BioRxiv) ROBO4 was identified as binding partner for ARHGAP39. Motivated by the possible interaction of mammalian ARHGAP39 with all human ROBO receptors, I aimed to functionally characterize ARHGAP39 and to investigate its function in guided cell migration. Together, my studies yielded insights into the role of ARHGAP39 in cell motility, both in randomly migrating cells and in SLIT2/ROBO-dependent guided migration, and provide the framework for future mechanistic studies.