Owing to their ability to reliably detect even very rare antigen-specific B cells in cellular isolates such as peripheral blood mononuclear cells (PBMC), and doing so robustly in a high throughput-compatible manner, B cell ELISPOT/FluoroSpot (collectively "B cell ImmunoSpot") tests have become increasingly attractive for immune monitoring in regulated settings. Presently, there are no guidelines for the qualification and validation of B cell ImmunoSpot assay results. Here, we propose such guidelines, building on the experience acquired from T cell ImmunoSpot testing in an environment adhering to the requirements of regulatory bodies yet taking the unique features of B cell assays into account. A streamlined protocol is proposed that permits the performance of all tests needed for the formal validation of an antigen-specific B cell ImmunoSpot assay in only three experiments, utilizing 2.2 × 10