Intestinal permeability is an important diagnostic marker, yet its determination by established tests, which measure the urinary excretion of orally administered tracer molecules, is time consuming and can only be performed prospectively. Here, we aim to validate proposed surrogate biomarkers, which allow measuring intestinal permeability more easily. In this cross-sectional study, we included two independent cohorts comprising nonobese (Healthy cohort, n = 51) and individuals with obesity (Obesity cohort, n = 27). The lactulose/mannitol (lac/man) ratio was determined in all individuals as an established marker of intestinal permeability. Furthermore, we measured six potential surrogate biomarkers, being albumin, calprotectin, and zonulin, measured in feces, as well as intestinal fatty acid binding protein (I-FABP), lipopolysaccharide binding protein (LBP) and zonulin, measured in plasma. Correlation analyses and multiple linear regression models were conducted to assess possible associations between the established lac/man ratio and the proposed biomarkers by also evaluating a potential effect of age, body mass index (BMI), and sex. The lac/man ratio correlated with plasma LBP levels in all cohorts consistently and with the amount of fecal zonulin in overweight and obese individuals. Multiple linear regression models showed that the association between the lac/man ratio and plasma LBP was independent of age, BMI, and sex. Fecal zonulin levels were associated with the lac/man ratio as well as BMI, but not age and sex. Our data suggest plasma LBP as a promising biomarker for intestinal permeability in adults and fecal zonulin as a potential biomarker in overweight and obese individuals.NEW & NOTEWORTHY This study shows that biomarkers from blood and fecal samples are associated with the cumbersome established tests of intestinal permeability throughout different cohorts. Therefore, such biomarkers could be used to assess gut barrier function in prospective cohort studies and large-scale clinical trials for which tracer-based tests may not be feasible.