The direct protein profiling of mammalian cells and bacteria has a growing influence in biotechnology as a high information bearing method for characterization of cells and cell states. Monitoring of proteins excreted in culture media not only serves to produce data on product yield and quality but provides important information on cell viability and nutrient supply that forms the basis for future process and expression optimization. Fast and simple MALDI mass spectrometry approaches were developed to efficiently characterize such complex biological systems. Several mammalian cell lines including CHO DXB11, CHOSSF3, and hybridomas were investigated; the lysis process, the sample pretreatment, and the matrix preparation were optimized for MALDI conditions. Initial experiments to observe the success of protein translation in gene expression experiments were performed. Using MALDI-compatible detergents, it was possible to extend the mass range detectable by MALDI mass spectrometry from the current range of 16 000 to 75 000 Da. In this mass range, the data are complementary (offering a better mass accuracy) to those obtained by SDS−PAGE electrophoresis experiments. These new methods were used to monitor a large-scale cultivation of hybridoma cells expressing an antibody of the IgG type. The increase in whole antibody and antibody light-chain protein, 8650 Da, and the decrease of insulin were followed during the monitoring period. Quantitative measurements of the IgG level during the cultivation compared favorably with those obtained by affinity HPLC.