Practical solutions for multiple antibiotic determination in food is required by the food industry and regulators for cost effective screening purposes. This study describes the feasibility in development and preliminary performance of a novel multispot nanoarray for antibiotic screening in honey. Using a multiplex approach the metabolites of the four main nitrofuran antibiotics including morpholinomethyl-2-oxazolidone (AMOZ), 3-amino-2-oxazolidinone (AOZ), semicarbazide (SEM), 1-aminohydantoin (AHD) and chloramphenicol (CAP) were simultaneously detected. Antibodies specific to the five antibiotics were nano-spotted onto microtitre plate wells and a direct competitive assay format was employed. The assay characteristics and performance were evaluated for feasibility as a screening tool for antibiotic determination in honey to replace traditional ELISAs. Optimisation of the spotting and assay parameters were undertaken with both individual and multiplex calibration curves generated in PBS buffer and a honey matrix. The limits of detection as determined by the 20% inhibitory concentrations (IC20) were determined as 0.19, 0.83, 0.09, 15.2 and 35.9 ng/mL in PBS buffer, 0.34, 0.87, 0.17, 42.1 and 90.7 ng/mL in honey (fortified at the start of the extraction) and 0.23, 0.98, 0.24, 24.8 and 58.9 ng/mL in honey (fortified at the end of the extraction) for AMOZ, AOZ, CAP, SEM and AHD respectively. This work has demonstrated the potential of multiplex analysis for antibiotics with results available for 40 samples within a 90 min period for antibiotics sharing a common sample preparation. Although both the SEM and AHD assay do not show the required sensitivity with the antibodies available for use to meet regulatory limits, with further improvements in these particular antibodies this multiplex format has the potential to show a reduction in cost with reduced labour time in combination with the high throughput screening of samples. This is the first 96 well spotted microtitre plate nanoarray for the semi-quantitative and simultaneous analysis of antibiotics.