Proteomic analysis of limited samples and single cells requires specialized methods that prioritize high sensitivity and minimize sample loss. Consequently, sample preparation is one of the most important steps in limited sample analysis workflows to prevent sample loss. In this work, we have eliminated sample handling and transfer steps by processing intact cells directly in the separation capillary, online with capillary electrophoresis coupled to tandem mass spectrometry (CE-MS/MS) for top-down proteomic (TDP) analysis of low numbers of mammalian cancer cells (<10) and single cells. We assessed spray voltage injection of intact cells from a droplet of cell suspension (∼1000 cells) and demonstrated 0−9 intact cells injected with a dependency on the duration of spray voltage application. Spray voltage applied for 2 min injected an average of 7 ± 2 cells and resulted in 33−57 protein and 40−88 proteoform identifications (N = 4). To analyze single cells, manual cell loading by hydrodynamic pressure was used. Replicates of single HeLa cells (N = 4) lysed on the capillary and analyzed by CE-MS/MS demonstrated a range of 17−40 proteins and 23−50 proteoforms identified. An additional cell line, THP-1, was analyzed at the single-cell level, and proteoform abundances were compared to show the capabilities of single-cell TDP (SC-TDP) for assessing cellular heterogeneity. This study demonstrates the initial application of TDP in single-cell proteome-level profiling. These results represent the highest reported identifications from TDP analysis of a single HeLa cell and prove the tremendous potential for CE-MS/MS on-capillary sample processing for high sensitivity analysis of single cells and limited samples.