Background: Serum biomarkers of steatosis such as the SteatoTest are recommended for large-scale screening studies, because imaging is less accessible and more expensive. Aims The primary aim of this retrospective analysis of prospective studies was to construct a new SteatoTest-2 that was not inferior to the reference first-generation SteatoTest, but that did not include BMI or bilirubin, as these two components can increase test variability because of the assessment of weight and height and in case of Gilbert syndrome or hemolysis, respectively. Patients and methods Five different subsets of 2997 patients with biopsies were evaluated for test construction and validation, and four to assess the prevalence of steatosis in target populations with increasing risks of steatosis. The performance of the SteatoTest-2 was compared with the reference test, using the noninferiority test (0.10 margin) and the Lin concordance coefficient. Results: Areas under the receiver operating characteristic curve of the SteatoTest-2 were noninferior to the reference test (P <0.001). Areas under the receiver operating characteristic curve varied in the SteatoTest-2 and the reference test according to subsets and the prevalence of steatosis, with 0.772 [95% confidence interval (CI): 0.713–0.820] versus 0.786 (95% CI: 0.729–0.832) in the 2997 cases with biopsy and 0.822 (95% CI: 0.810–0.834) versus 0.868 (95% CI: 0.858–0.878) in the 5776 cases including healthy individuals without risk factors of steatosis as controls, respectively. The Lin coefficient was highly concordant (P< 0.001), from 0.74 (95% CI: 0.74–0.74) in presumed NAFLD to 0.91 (95% CI: 0.89–0.93) in the construction subset. Conclusion: The SteatoTest-2 is simpler and noninferior to the first-generation SteatoTest for the diagnosis of steatosis, without the limitations of BMI and bilirubin.