The junctional sarcoplasmic reticulum (jSR) is a critical organelle in cardiomyocytes, regulating calcium homeostasis and Excitation–Contraction Coupling (ECC). A quantitative understanding of its protein composition is essential for investigating cardiac physiology and related pathologies. However, isolating intact jSR vesicles, particularly those enriched in membrane proteins, remains a challenging task. Here, we describe our optimized protocol for reproducible enrichment of jSR vesicles from a single murine heart, without the use of antibodies. The protocol enables the recovery of low-abundance membrane proteins while preserving their native interactions with partners. This strategy facilitates the straightforward identification by Mass Spectrometry of highly relevant yet challenging jSR proteins, including the cardiac Ryanodine Receptor and calsequestrin. Our protocol provides a robust tool for studying the structural and stoichiometric organization of the cardiac jSR components in a widely used animal model.