Sensing of nucleic acids (NAs) by Toll-like receptors (TLRs) is crucial in the host defence against viruses and bacteria. Yet, these TLRs can contribute to autoimmunity. UNC93B1 is a key-regulator of NA-sensing TLRs by controlling the translocation of TLRs from the ER into endolysosomes. Without functional UNC93B1, NA-sensing TLRs are retained in the ER, incapable of ligand recognition. Patients lacking UNC93B1 show defective signalling of endosomal TLRs and are at increased risk to develop herpes simplex virus encephalitis. To date, molecular insights into UNC93B1-mediated regulation of human NA-sensing TLRs are lacking. In this study, we aimed to investigate the role of trafficking motifs and post-translational modifications of UNC93B1 for human endosomal TLR activity. To this end, we utilised mass spectrometry in order to identify post-translational modification sites and prepared mutant forms of UNC93B1 by site-directed mutagenesis. EBV immortalized B cells from UNC93B1-deficient patients as well as UNC93B1 KO THP1s generated by CRISPR-Cas-based genome editing were reconstituted with wild type or mutant forms of UNC93B1 and assessed for endosomal TLR activity. The localization of UNC93B1 was analysed by confocal microscopy. The destruction of either trafficking motifs or post-translational modification sites resulted in receptor-, ligand-, and cell-type specific defects of human endosomal TLRs. Furthermore, mass spectrometry revealed that wild type versus mutant forms of UNC93B1 differed in terms of interaction partners and cellular compartment signatures. Our study reveals another level of complexity of UNC93B1-mediated TLR regulation and thereby contributes to the understanding of nucleic-acid sensing TLR biology.