Shedding of circulating tumor cells (CTCs) from solid tumors into the bloodstream contributes to metastasis and is estimated to be responsible for 90 % of cancer-related deaths. The scarcity of these cells makes their isolation and analysis a particular challenge. Until now, genomic and transcriptomic methodologies have characterized these rare cells, revealing great cellular heterogeneity. However, proteomic analyses of CTCs have only recently become in reach due to recent advances in ultrasensitive mass spectrometry based proteomics. Gaining comprehensive proteomics data from CTCs is essential for a holistic understanding of the mechanisms of metastasis, comprehensive cell characterization, and the identification of novel treatment targets. We established a first-of-its-kind microscopy-guided ultrasensitive LC-MS workflow to detect and profile CTCs. The combination of microfluidic cell enrichment, cytospin centrifugation with immunofluorescent staining and high-resolution imaging enables optimal differentiation of tumor cell phenotypes and lymphocytes. Using a cancer-specific antibody panel allowed us to identify various tumor subpopulations to capture different cellular states undergoing epithelial-mesenchymal transition (EMT). This approach goes beyond assessing only epithelial cells, which is insufficient to capture high phenotypic tumor heterogeneity. Further, we combined imaging data with the deep visual proteomics workflow employing single-cell laser microdissection followed by robotic sample preparation and mass-spectrometry based low-input proteomics. We will apply our unique workflow to analyze CTCs from liquid biopsies from patients with locally advanced head and neck squamous cell carcinoma. Studying molecular properties on the protein level will supplement and expand the knowledge about CTCs almost exclusively from genomic and transcriptomic perspectives to unveil valuable means of metastatic mechanisms. We anticipate that our findings will provide valuable insights into disease progression and may have implications for the development of novel targeted therapies for anti-metastatic cancer treatment.