Abstract: Proteomic analysis has become a cornerstone of modern biomedical research, providing critical insights into physiological and pathological processes within biological systems, particularly in molecular diagnostics. The construction of proteomic profiles of cancer cells holds significant importance for understanding disease mechanisms, diagnosis, and treatment. Due to cellular heterogeneity, the study of small-scale and single-cell samples has emerged as a persistent focus in proteomics, while the application of proteomics in biomedicine faces several challenges, including the scarcity of samples, low efficiency of sample preparation, lengthy workflows, low throughput, and significant sample loss. However, significant advancements have been made in proteomic research technologies recently. Firstly, instrument manufacturers have introduced innovative techniques and instruments with enhanced sensitivity, providing scientists with advanced tools that have greatly facilitated the feasibility of proteomic analysis for small-scale and single-cell samples. Secondly, the development of rapid and integrated platforms has helped to shorten workflows and increase throughput. Among all the studies, micro- and nanoscale techniques have the potential to overcome the challenges mentioned above. In this study, the matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) were combined to explore the application of functionalized micro-enzyme reactors in the rapid analysis of trace amount of live cell line proteomes, and a comprehensive evaluation of their analytical performance was provided. Efficient cell lysis and protein extraction are achieved by encapsulating the functionalized enzymatic micro-reactors in pipette tips and combining guanidine hydrochloride with a dual-enzyme system (trypsin and DNase I). The experimental results demonstrated that this method enables efficient online cell lysis, protein extraction, and digestion for trace cell samples. Notably, when processing 1×10⁴ cells, the protein extraction efficiency is significantly superior to that of the traditional filter-aided sample preparation (FASP) method. Moreover, the reaction time for live cell samples in the enzymatic micro-reactor is only 6 min, and the entire analysis process is completed in less than 1 h, highlighting the potential of this functionalized micro- enzymatic reactor system for high-throughput online sample preparation. This study provides a new technological platform for the rapid high-throughput proteomic analysis of trace live cell lines. Integrating MALDI-MS and UHPLC-MS/MS enables a comprehensive evaluation and application of proteomics sample preparation method.