The human CXC chemokine receptor 1 (CXCR1), a G protein-coupled receptor (GPCR), plays significant roles in inflammatory diseases and cancer. While CXCL8 is a well-established high-affinity ligand for CXCR1, there is no consensus regarding the binding ability of the other ELR+ chemokines (CXCL1-3 and CXCL5-8). Since research has predominantly focused on CXCL8-mediated CXCR1 signaling, insight into potential signaling bias induced by different CXCR1 ligands is lacking. Therefore, in this study we first compared and clarified the binding ability of all ELR+ chemokines using a competition binding assay. In this assay CXCL1-3 and CXCL5 behaved as low-affinity ligands while CXCL6-8 were high affinity ligands. We further investigated potential ligand bias within the CXCR1 signaling system. Using NanoBRET-based assays heterotrimeric G protein dissociation, β-arrestin recruitment and receptor internalization induced by chemokine binding to CXCR1 were investigated. A quantitative and qualitative investigation of ligand bias showed that the high-affinity ELR+ chemokines were biased towards G protein activation over β-arrestin recruitment and receptor internalization, when CXCL8 was used as a reference ligand.