Studies have suggested that cancerous tissue has a lower ¹⁵ N/ ¹⁴ N ratio than benign tissue. However, human data have been inconclusive, possibly due to constraints on experimental design. Here, we used high-sensitivity nitrogen isotope methods to assess the ¹⁵ N/ ¹⁴ N ratio of human breast, lung, and kidney cancer tissue at unprecedented spatial resolution. In lung, breast, and urothelial carcinoma, ¹⁵ N/ ¹⁴ N was negatively correlated with tumor cell density. The magnitude of ¹⁵ N depletion for a given tumor cell density was consistent across different types of lung cancer, ductal in situ and invasive breast carcinoma, and urothelial carcinoma, suggesting similar elevations in the anabolism-to-catabolism ratio. However, tumor ¹⁵ N depletion was higher in a more aggressive metaplastic breast carcinoma. These findings may indicate the ability of certain cancers to more effectively channel N towards growth. Our results support ¹⁵ N/ ¹⁴ N analysis as a potential tool for screening biopsies and assessing N metabolism in tumor cells.