Dengue virus (DENV) is a Flavivirus of the Flaviridae family of (+)RNA viruses that causes significant morbidity and mortality in tropical and sub-tropical areas worldwide. A major determinant of the vascular leakage that is associated with severe DENV infections is the viral non-structural protein NS1. In addition to its essential roles in viral RNA replication and infectious virus production, NS1 is secreted from infected cells as a hexameric lipoparticle and it is this secreted form of the protein that can induce vascular leakage via induction of inflammatory cytokine production and endothelial glycocalyx disruption. Despite the importance of NS1 secretion in DENV pathogenesis, the exact features of NS1 that are critical to its secretion from infected cells are not fully characterised. Here we employed random point mutagenesis and luminescent peptide (HiBiT)-tagged NS1 expression constructs to identify NS1 residues that are critical to its secretion. Amongst others, multiple individual mutations in the ß-ladder domain of NS1 (including V220D, A247V, T283A, C313S and R336S) impaired its secretion by >90%. The impact of these mutations on viral RNA replication and infectious virus production are currently under investigation. Together, we suggest that residues that are critical for NS1 secretion may be targeted in future antiviral drug and attenuated vaccine development.