Introduction Basophil activation test (BAT) and immunoassays are the most widely used in vitro tests to diagnose IgE-mediated allergic reactions to penicillin. However, studies to determine if one test is interdependent from another are limited. Objective The present study aimed to measure the agreement between BAT and immunoassay in diagnosis of penicillin allergy. Method BAT was performed using penicillin G (Pen G), penicillin V (Pen V), penicilloyl-polylysine (PPL), minor determinant mix (MDM), amoxicillin (Amx) and ampicillin (Amp) in 25 patients. Immunoassay of total IgE (tIgE) and specific IgE (sIgE) antibodies to Pen G, Pen V, Amx and Amp were quantified. Skin prick test (SPT) using PPL-MDM, Amx, Amp and Clavulanic acid were also performed. Results Minimal agreement was observed between BAT and immunoassay (k = 0.25). Of two BAT-positive patients, one patient is positive to Amx (59.27%, SI = 59) and Amp (82.32%, SI = 82) but sIgE-negative to all drug tested. This patient is also SPT-positive to both drugs. Another patient is BAT-positive to Pen G (10.18%, SI = 40), Pen V (25.07%, SI = 100) and Amp (19.52%, SI = 79). In sIgE immunoassay, four patients were sIgE-positive to at least one of the drugs tested. The sIgE level of three patients was between low and moderate and they were BAT-negative. One BAT-positive patient had a high level of sIgE antibodies (3.50–17.5 kU/L) along with relatively high specific to total IgE ratio ≥0.002 (0.004–0.007). Conclusions The agreement between BAT and immunoassay is minimal. Performing both tests provides little increase in the sensitivity of allergy diagnosis work-up for immediate reactions to penicillin.