Proteomics analysis with large number of cells represents the average protein content of a heterogenous mixtures of cells. Understanding heterogeneity at single cell level is of great interest for biomedical research. MS-based proteomics is a promising technique for single cell analysis by enabling identification and quantification of thousands of proteins in unbiased manner. However, due to inefficient single cell isolation, large sample losses during sample preparation and low throughput, the extension to single cell studies has been largely ineffective. To address these challenges, we combined nanoPOTS (Nanodroplet Processing in One-pot for Trace Samples) technology with tandem mass tag (TMTTM) isobaric labeling to efficiently process and analyze single mammalian cells containing <0.2 ng total proteins on new Orbitrap Eclipse™ Tribrid™ Mass Spectrometers with real time search and FAIMS Pro™ to improve single cell proteome coverage and enhance quantification accuracy. Single cells were isolated from cultured murine and HeLa cells via fluorescence-activated cell sorting and samples were processed on nanoPOTS chip. The UltiMate™ 3000 RSLCnano system was used with 20 to 30 µm i.d. columns coupled to Orbitrap Eclipse. Proteome Discoverer™ 2.4 software was used for data analysis. Both label-free and isobaric labelling (TMT10plex™)-based protein quantifications were evaluated with a focus on reproducibility in quantification and throughput. The label-free workflow resulted in identification of an average of 465 protein groups from single Hela cells and 802 protein groups from a large HeLa cell with high-confidence MS/MS spectra. The TMT10plex analysis of the three cultured murine cell populations (C10, SVEC and Raw cells) enabled identification of 2346 proteins and 1300 quantifiable among 40 single cells. We have demonstrated that single cell proteome can be quantified using label-free or TMT workflows by combing nanoPOTS with Orbitrap Eclipse Tribrid mass spectrometer, enabling researchers to investigate cell heterogeneity as well as rare cells.