Vitamin A (retinol) is essential in key biological processes such as embryonic development, immune response, and nervous system function. All-trans-retinoic acid (atRA), an active vitamin A metabolite, requires precise metabolic regulation as an excess or deficiency can lead to serious health defects. atRA homeostasis is regulated by a variety of binding proteins and enzymes, such as the short-chain dehydrogenase (SDR) enzymes. The SDR enzymes retinol dehydrogenase 10 (RDH10) and short-chain dehydrogenase reductase 3 (DHRS3) compose the antagonistically bifunctional retinoid oxidoreductase complex (ROC), one mechanism by which RA is regulated. RDH10 oxidizes retinol (ROL) to retinaldehyde (RAL) whereas DHRS3 reduces RAL back to ROL. Retinaldehyde dehydrogenase (RALDH) then oxidizes RAL to atRA. Elevated levels of atRA are known to lead to congenital defects such as craniosynostosis. Mutations in DHRS3 have been identified in craniosynostosis patients using whole exome sequencing (WES), whole genome sequencing (WGS), and single nucleotide polymorphism (SNP) arrays. These mutations, while not in the active site, are highly conserved and result in DHRS3 loss of function. This thesis focuses on the characterization of the impact of these human mutations in DHRS3 on DHRS3 enzymatic activity, DHRS3 protein half-life, and on the global proteomic profile. To determine the impact of mutations on reductive activity of DHRS3, DHRS3 wild-type (WT) and mutants were stably transfected in HEK293 cells. When treated with 1 uM RAL, cell lines expressing the DHRS3 mutants had significantly higher levels of atRA compared to the DHRS3 WT consistent with a loss of function. Co-transfection of RDH10 along with DHRS3 investigated if mutations effected the ability of DHRS3 and RDH10 to activate and stabilize each other. In the co-transfected lines, RDH was able to exacerbate the production of atRA in DHRS3 mutant cell lines due to its unopposed oxidation of ROL to RAL. To determine the effect of DHRS3 mutations on protein half-life, a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed, using a DHRS3-specific peptide and parallel reaction monitoring to quantify DHRS3 protein levels. Using stable isotope labeling by amino acids in cell culture (SILAC), the half-life of DHRS3 protein for each of the DHRS3 mutant variants was calculated. Lastly, to determine the impact of each DHRS3 variant on the global proteome due to atRA dysregulation, untargeted differential expression analysis using LC-MS/MS was performed; changes to individual proteins as well as canonical pathways were determined. Together these studies will advance our understanding of how DHRS3 mutations contribute to dysregulation of retinoid homeostasis as well as the proteome which will contribute to the elucidation of mechanisms by which DHRS3 mutations contribute to atRA-induced craniosynostosis.