Neurofibromatosis type 1 (NF1) and Legius syndrome (LS) are caused by inactivating variants in NF1 and SPRED1. NF1 encodes neurofibromin (NF), a GTPase activating protein (GAP) for RAS, that interacts with the SPRED1 product, Sprouty-related protein with an EVH (Ena/Vasp homology) domain 1 (SPRED1). Establishing a clinical and molecular diagnosis of NF1 or LS can be challenging due to the phenotypic diversity, the size and complexity of the NF1 and SPRED1 loci and uncertainty over the effects of variants on pre-mRNA splicing and NF/SPRED1 function. The purpose of this work was to improve NF1 and SPRED1 variant classification. To help establish the pathogenicity of NF1 and SPRED1 variants identified in individuals with NF1 or LS, we employed 4 assays: (i) analysis of patient RNA by RT-PCR; (ii) in vitro exon trap analysis of NF1 pre-mRNA splicing; (iii) in vitro analysis of NF RAS GAP activity; and (iv) in vitro analysis of the NF-SPRED1 interaction. In 69/105 (66%) cases we obtained evidence to support variant pathogenicity according to American College of Medical Genetics guidelines, demonstrating the utility of functional approaches for NF1 and SPRED1 variant classification and NF and LS diagnostics.