Background/Objectives: N-terminal-proBNP (NT-proBNP) is a biomarker released into the blood in response to heart failure, reflecting the extent of cardiac stress and damage. QuidelOrtho Diagnostics released its latest reformulation of its NT-proBNP assay, the Vitros NT-proBNP II assay. This study aims to evaluate the analytical performance of the Vitros NT-proBNP II assay. Methods: Repeatability, reproducibility, carryover, analytical measurement range, and clinical reportable range (AMR and CRR) were assessed using commercially available materials and dilution of patient specimens. Accuracy was evaluated by comparing results from the Vitros NT-proBNP II and the Vitros NT-proBNP assays. Paired heparin and EDTA plasma specimen results were compared, and instrument-to-instrument comparison was performed using two different Vitros 5600 Integrated Systems. NT-proBNP stability was evaluated at room temperature, 2–8 °C, and −18 °C for up to five days. Results: Repeatability and reproducibility were ≤10% CV, and no carryover was observed. The AMR was 20–30,000 pg/mL and dilution up to 80 times was verified. Passing–Bablok analysis showed a significant proportional bias with a slope of 1.37. Instrument-to-instrument and heparin-to-EDTA plasma comparisons showed no significant biases. NT-proBNP is stable up to five days at room temperature, 4 °C, and −20 °C. Conclusions: Our evaluation demonstrated acceptable analytical performances of the Vitros NT-proBNP II assay except for the positive proportional bias compared with the Vitros NT-proBNP assay.