BACKGROUND: The incorporation of monoclonal antibodies, such as daratumumab, into multiple myeloma treatment regimens has led to the issue of false-positive interference in both serum protein electrophoresis (SPEP) and immunofixation (IF). The Hydrashift assay removes daratumumab interference from IF, allowing for correct interpretation. Here, we retrospectively examined the use of the Hydrashift assay at a large cancer center and provide guidelines on its most appropriate use. METHODS: 38 patients with distinct daratumumab peaks on their SPEP were selected and were used to quantify the daratumumab peak on SPEP using the Sebia Phoresis software. A retrospective review of all Hydrashift assays ordered at our institution from July 2018 to March 2020 was performed. Data collected included patient clone type, IF migration patterns, and Hydrashift result. Serial quantification of SPEP results was performed as the corresponding IF transitioned from a true positive to a false positive. RESULTS: Daratumumab adds a maximum magnitude of 0.20 g/dL on SPEP. Serial SPEP quantification showed IF transitioned from true positive to false positive when M-spikes ranged from 0.09 g/dL to 0.11 g/dL. Over 20 months, our laboratory performed 280 Hydrashift assays on 96 patients, 43/96 of whom had comigrating daratumumab/IgG-K IF bands. CONCLUSIONS: The Hydrashift assay is typically unnecessary in patients with large M-spikes, >0.25 g/dL, regardless of clone type. When patient history is available, we recommend the Hydrashift assay be used in patients with comigrating daratumumab/IgG-K bands with M-spikes of <0.25 g/dL.