Toward a better measure of functional laterality: Comparing and refining laterality indices in resting-state functional connectivity

• Methodological rigor in laterality assessment This study provides the first systematic benchmarking of sign-handling schemes in resting-state fMRI laterality metrics. By clarifying how analytic decisions shape hemispheric asymmetries, we establish a methodological foundation that improves transparency, reproducibility, and interpretability in laterality research. • Novel conceptual framework for asymmetry We introduce a threshold-aware metric that jointly captures hemispheric segregation and inter-hemispheric integration. This framework extends beyond traditional laterality measures, enabling a more nuanced characterization of brain asymmetry and offering a new tool for studying the balance between specialization and communication. • Cognitive relevance of lateralization Our analyses reveal that left temporal segregation is associated with vocabulary skills, while right ventral temporal–parietal integration supports visuospatial performance. These findings link lateralized functional organization to domain-specific cognition, advancing understanding of how hemispheric specialization underpins human abilities. • Implications for neuroscientific models We demonstrate that negative-only connectivity approaches obscure integration clusters under strict thresholds, cautioning against oversimplified analytic practices. Our results emphasize balanced treatment of connectivity signs, with broader implications for modeling hemispheric specialization in both healthy and clinical populations. Systematic investigations into the lateralized human brain have revealed a bivariate functional architecture that underpins distinct cognitive processes. This architecture manifests through inter- and intra-hemispheric lateralization, captured respectively by neural integration and segregation. In this study, we conducted a comprehensive evaluation of multiple quantitative laterality metrics in resting-state fMRI connectivity, using conceptual models to illustrate how inter- and intra-hemispheric correlations shape functional lateralization. We further highlight the critical influence of factors such as correlation sign, correlation coefficient distribution, and statistical thresholding methodology on the interpretation of functional connectivity-based laterality indices. Our findings show that, in our dataset, laterality metrics based on positive-only functional connectivity with a lenient connection-level threshold most consistently capture established relationships between functional brain lateralization and performance in language and visuospatial domains.