Background: Bullous pemphigoid (BP) is an autoimmune blistering disease associated with autoantibodies against BP180 and/or BP230 antigens. The immunoassays available for serological diagnostics include indirect immunofluorescence (IIF) on monkey esophagus (ME), salt‐split skin (SSS), and enzyme‐linked immunosorbent assay (ELISA) for BP180‐NC16a and BP230. Only a few studies validated innovative BIOCHIP mosaic, but none compared agreement between BIOCHIP substrates with conventional methods separately. Methods: We evaluated the agreement between BIOCHIP and conventional methods and assessed sensitivity and specificity in BP diagnosis. The study comprised 51 BP patients and 39 controls. Results: Analysis showed very good agreement between BIOCHIP‐SSS vs classic IIF‐SSS (0.933, P < 0.001) and for BIOCHIP‐BP180‐NC16a vs ELISA‐BP180‐NC16a (0.933, P < 0.001). A good strength of agreement between BIOCHIP‐ME vs classic IIF‐ME was observed (0.694, P < 0.001) similar to BIOCHIP‐BP230 vs ELISA‐BP230 (0.793, P < 0.001). BIOCHIP‐ME sensitivity was 51.0%, whereas IIF‐ME was 76.5%. Epidermal reaction on BIOCHIP‐SSS was found in 94.1% of BP patients and in all patients on IIF‐SSS (sensitivity 100%). BIOCHIP‐BP180‐NC16a sensitivity was lower than in ELISA‐BP180‐NC16a (76.5% vs 82.4%). BP230 sensitivity of both methods was similar (45.1% vs 43.1%). The specificity for all antigens was 100%. Conclusion: BIOCHIP mosaic is a useful method presenting satisfactory agreement with conventional immunoassays. [ABSTRACT FROM AUTHOR]