Collagen is the major component of the extracellular matrix in mammals and its characteristics provide important information about the state of connective tissue. There are only few methods of label-free visualization of collagen fibers; the most frequently used is the second harmonic generation (SHG) microscopy. SHG microscopy is a non-invasive technique for the assessment of the abundance and structure of fibrillar collagen with a high resolution and specificity. At constant measurement parameters (magnification, excitation power, resolution, digital gain of registration matrix), quantitative analysis of SHG images provides a reliable characterization of collagen state. Current approaches to the SHG signal quantification are numerous and typically should be adapted to a specific task. In this review, we systematize the variety of these approaches and present the examples of biomedical application of the SHG signal quantitative analysis, as well of combined application of SHG and autofluorescence imaging.