This article introduces a novel single-cell proteomic analysis method called PiSPA, which enables the quantification of up to 3000 protein groups in a mammalian cell. The method uses a label-free quantitative approach and is based on a nanoliter-scale microfluidic liquid handling robot, which allows for the capture, pretreatment, and injection of single cells. The PiSPA workflow was tested on three types of mammalian cells (A549, HeLa, and U2OS) and achieved quantification of 2449–3500, 2278–3257, and 1621–2904 protein groups, respectively. The method also enables the study of HeLa cell migration at the single-cell proteome level, demonstrating its potential for practical biological research. The article discusses the challenges of single-cell proteomic analysis, including the low protein content in single cells and the difficulty of amplifying proteins as nucleic acids. It reviews various single-cell proteomic techniques, including those based on specific antibody labeling and non-labeling proteome analysis with high-resolution mass spectrometry. The article highlights the limitations of current methods and the need for improved identification depth and simplified devices and operations to achieve practical whole-process proteomic analysis at the single-cell level. The PiSPA platform integrates a probe-based microfluidic liquid handling robot with a commercial liquid chromatograph and a trapped ion mobility spectrometry (TIMS) QTOF mass spectrometer. The platform enables automated pick-up operation mode for cell sorting and pretreatment, including the sorting of single cells, multi-step pretreatment, and injection of peptide samples to the LC column. The platform also utilizes a single-cell customized strategy that considers the effects of the unique properties of single cells on sample pretreatment, separation, and detection. The article presents results from the PiSPA platform, including the quantification of protein groups in single cells under different modes (DDA and DIA). The results show that the PiSPA platform can achieve high protein identification numbers and good repeatability. The platform was also used to study the proteomic analysis of migrating HeLa cells, revealing significant differences in protein expression between migrated and control cells. The article discusses the advantages of the PiSPA platform, including its ability to achieve high protein identification numbers, good repeatability, and the potential for practical application in single-cell proteomic analysis. The platform's integration of microfluidic technology and automated operations makes it a promising tool for single-cell proteomic analysis. The article concludes that the PiSPA platform has the potential to reveal the intrinsic control factors behind apparent behaviors in single-cell experiments and could provide an effective tool for cell migration studies and the development of anticancer approaches and drugs.This article introduces a novel single-cell proteomic analysis method called PiSPA, which enables the quantification of up to 3000 protein groups in a mammalian cell. The method uses a label-free quantitative approach and is based on a nanoliter-scale microfluidic liquid handling robot, which allows for the capture, pretreatment, and injection of single cells. The PiSPA workflow was tested on three types of mammalian cells (A549, HeLa, and U2OS) and achieved quantification of 2449–3500, 2278–3257, and 1621–2904 protein groups, respectively. The method also enables the study of HeLa cell migration at the single-cell proteome level, demonstrating its potential for practical biological research. The article discusses the challenges of single-cell proteomic analysis, including the low protein content in single cells and the difficulty of amplifying proteins as nucleic acids. It reviews various single-cell proteomic techniques, including those based on specific antibody labeling and non-labeling proteome analysis with high-resolution mass spectrometry. The article highlights the limitations of current methods and the need for improved identification depth and simplified devices and operations to achieve practical whole-process proteomic analysis at the single-cell level. The PiSPA platform integrates a probe-based microfluidic liquid handling robot with a commercial liquid chromatograph and a trapped ion mobility spectrometry (TIMS) QTOF mass spectrometer. The platform enables automated pick-up operation mode for cell sorting and pretreatment, including the sorting of single cells, multi-step pretreatment, and injection of peptide samples to the LC column. The platform also utilizes a single-cell customized strategy that considers the effects of the unique properties of single cells on sample pretreatment, separation, and detection. The article presents results from the PiSPA platform, including the quantification of protein groups in single cells under different modes (DDA and DIA). The results show that the PiSPA platform can achieve high protein identification numbers and good repeatability. The platform was also used to study the proteomic analysis of migrating HeLa cells, revealing significant differences in protein expression between migrated and control cells. The article discusses the advantages of the PiSPA platform, including its ability to achieve high protein identification numbers, good repeatability, and the potential for practical application in single-cell proteomic analysis. The platform's integration of microfluidic technology and automated operations makes it a promising tool for single-cell proteomic analysis. The article concludes that the PiSPA platform has the potential to reveal the intrinsic control factors behind apparent behaviors in single-cell experiments and could provide an effective tool for cell migration studies and the development of anticancer approaches and drugs.