question: OBJECTIVES: Studies investigating the association between vitamin D and severity of COVID-19 have mixed results perhaps due to immunoassay assessment of total 25-hydroxyvitamin D (tD) (the sum of 25-hydroxyvitamin-D2 [25-OH-D2] and 25-hydroxyvitamin-D3 [25-OH-D3]). Liquid chromatography tandem mass spectrometry (LC-MS/MS) has high analytical specificity and sensitivity for 25-OH-D2 and 25-OH-D3, and thus enables a more accurate assessment of impact on COVID-19 outcomes. METHODS: We established reference intervals for 25-OH-D3 and tD using LC-MS/MS. 25-OH-D2, 25-OH-D3 and tD were quantitated for 88 COVID-19 positive and 122 COVID-19 negative specimens. Chi-square or Fisher’s exact tests were used to test associations in binary variables. T-Tests or Wilcoxon rank sum tests were used for continuous variables. Cox proportional hazards were used to test associations between 25-OH-D3 or tD levels and length of stay (LOS). For mortality and ventilation, logistic regression models were used. RESULTS: COVID-19 patients with deficient (<20 ng/mL) levels of 25-OH-D3 had significantly longer LOS by 15.3 days. COVID-19 P patients with deficient (<20 ng/mL) and insufficient (<30 ng/mL) of tD had significantly longer LOS by 12.1 and 8.2 days, respectively. Patients with insufficient levels of tD had significantly longer LOS by 13.7 days. COVID-19 patients with deficient serum 25-OH-D3 levels had significantly increased risk-adjusted odds of in-hospital mortality (OR [95% CI]: 5.29 [1.53–18.24]); those with insufficient 25-OH-D3 had significantly increased risk for requiring ventilation during hospitalization was found at LCMS insufficient cutoff (OR [95% CI]: 2.75 [1.10–6.90]). CONCLUSIONS: There is an inverse relationship of 25-hydroxyvitamin D levels and hospital LOS for COVID-19 patients. Vitamin D status is a predictor for severity of outcomes. LCMS results are useful for assessing the odds of mortality and the need for ventilation during hospitalization.