Autoantibody detection is a useful and obligatory tool for clinicians and researchers in diagnosing autoimmune diseases. Line blotting is a simple, sensitive, and flexible technique for fast semiquantitative detection of multiple antibodies. Line blotting enables the detection of antibodies on membrane strips coated with thin parallel lines of several purified, biochemically characterized antigens, which are fixed onto a synthetic support. Each strip can contain more than ten antigens, thus allowing simultaneous screening for multiple antibodies. Here, we describe the principle of line blotting and how the buffer composition can interfere with the results of autoantibody detection.