The paper describes on-line chromatographic separation and MS analysis of complex polypeptide mixture components by interfacing a high-performance microcolumn liquid chromatograph MiliChrom A-02 to an orthogonal electrospray ion source time-of-flight mass spectrometer MX5303. The potential of the tandem is demonstrated on the example of target- and by-product content monitoring at certain stages of the industrial genetic engineering-based production of human insulin.