Single-cell profiling provides insights into cellular behaviour that macroscale cell cultures and bulk measurements cannot reveal. In the context of personalized cancer treatment, the profiling of individual tumour cells may lead to higher success rates for therapies by rapidly selecting the most efficacious drugs. Currently, genomic analysis at the single-cell level is available through highly sensitive sequencing approaches. However, the identification and quantification of intracellular or secreted proteins or metabolites remains challenging. Here, we introduce a microfluidic method that facilitates capture, automated data acquisition and the multiplexed quantification of proteins from individual cells. The microfluidic platform comprises 1026 chambers with a volume of 152 pL each, in which single cells and barcoded beads are co-immobilized. We demonstrated multiplexed single-cell protein quantification with three different mammalian cell lines, including two model breast cancer cell lines. We established on-chip immunoassays for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), galectin-3 (Gal-3) and galectin-3 binding protein (Gal-3bp) with detection limits as low as 7.0 × 10⁴, 2.3 × 10⁵ and 1.8 × 10³ molecules per cell, respectively. The three investigated cell types had high cytosolic levels of GAPDH and could be clearly differentiated by their expression levels of Gal-3 and Gal-3bp, which are important factors that contribute to cancer metastasis. Because it employed commercially available barcoded beads for this study, our platform could be easily used for the single-cell protein profiling of several hundred different targets. Moreover, this versatile method is applicable to the analysis of bacteria, yeast and mammalian cells and nanometre-sized lipid vesicles.