N-Myc downregulated-like (NDL) proteins belong to the α/β-hydrolase-fold-containing protein family. NDLs are interacting partners of G-protein subunits which are involved in abiotic and biotic stress signaling mechanisms in plants. Three NDLs (NDL1, NDL2, and NDL3) have been identified in Arabidopsis thaliana. The NDL1 interactome reveals its interaction with numerous proteins involved in diverse plant functions, suggesting its role in various signaling mechanisms. The current study was designed to explore the level of conservation of NDL proteins across the plant kingdom to analyze protein structure for the conserved sites involved in protein–protein interactions. We analyzed NDL proteins from different plant groups for sequence conservation patterns through phylogenetic and motif analyses. Subsequently, homology-based models were built for NDLs and their selected interactors using MODELLER, and interaction sites were also analyzed using molecular docking. Overall, our study revealed sequence conservation within the NDL family and the presence of several conserved motifs across diverse plant groups. Additionally, docking analysis suggests that two specific regions, spanning positions 40–50 and 135–170 in the NDL1 structure, may serve as hotspots for various protein–protein interactions.