Natural rubber (NR) is a crucial raw material in the automotive industry, particularly for tyre manufacturing. Its unique molecular architecture, based on a cis-1,4-polyisoprene matrix, imparts the distinctive properties of both raw and cured rubber. However, the chemical composition of non-rubber components entrapped within this polymer matrix remains only partially understood. These non-isoprene constituents account for approximately 6% of the total mass and consist mainly of proteins at the ω-terminal end of the polyisoprene chain and phospholipids at the α-end. Interactions between these functional chain ends generate non-covalent associations that enhance molecular entanglement in the elastomeric network. Using high-resolution mass spectrometry, a detailed characterization of protein and lipid fractions of NR was achieved. Collectively, proteomic and lipidomic insights provide a comprehensive picture of NR’s molecular composition, offering valuable perspectives for the rational design of next-generation, high-performance rubber-based materials.