Dengue virus (DENV) is a Flavivirus of the Flaviviridae 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 10 identified point mutations, several mutations within the β-ladder domain of NS1 (V220D, A248V, T283A, C313S and R336S) heavily impaired its secretion by >90%. Follow-up studies on two of these mutants, V220D and A248V revealed they did not support viral RNA replication nor infectious virus production. Subsequent analyses of these mutants by confocal microscopy and immunoblotting in the context of a non-structural protein (NS1-NS5) expression system indicated that these mutations may disrupt NS1 processing or maturation events that are required for mutiple NS1 functions, including its secretion. We propose that disruption of NS1 secretion may represent a viable target of future antiviral drug and attenuated vaccine development and that our mutagenesis and luminescent detection approaches can be exploited towards these goals.