The proximal tubule contains the highest expression of angiotensinogen mRNA and protein within the kidney and plays a vital role in the renal renin-angiotensin system. To study the regulation of angiotensinogen expression in the kidney in more detail, the proximal tubule needs to be accurately isolated from the rest of the nephron and separated into its three segments. The purpose of this study was to design a novel protocol using specific markers for the separation of proximal tubule cells into the three proximal tubule segments and to determine angiotensinogen expression in each segment. Kidneys were removed from C57BL/6J mice. The proximal tubules were aspirated from region of a Percoll gradient solution of the appropriate density. The proximal tubule was then separated into its three segments using segment-specific membrane proteins, after which each segment was characterized by a different specific marker (sodium-glucose transporter 2 for Segment 1; carbonic anhydrase IV for Segment 2; ecto-adenosine triphosphatase for Segment 3). The isolation of proximal tubules into three segments was successful, and angiotensinogen mRNA in Segment 2 and 3 and angiotensinogen protein in all three segments were confirmed. This protocol will be helpful for future studies of the detailed mechanisms of the intrarenal renin-angiotensin system.