Mass spectrometry (MS)-based proteomics has become a powerful technology to quantify the entire complement of proteins in cells or tissues. Here, we review challenges and recent advances in the LC-MS-based analysis of minute protein amounts, down to the level of single cells. Application of this technology revealed that single-cell transcriptomes are dominated by stochastic noise due to the very low number of transcripts per cell, whereas the single-cell proteome appears to be complete. The spatial organization of cells in tissues can be studied by emerging technologies, including multiplexed imaging and spatial transcriptomics, which can now be combined with ultra-sensitive proteomics. Combined with high-content imaging, artificial intelligence and single-cell laser microdissection, MS-based proteomics provides an unbiased molecular readout close to the functional level. Potential applications range from basic biological questions to precision medicine.