Laser-Induced Breakdown Spectroscopy (LIBS) was examined as a novel method to provide the readout of microtiter plate immunoassays involving nanoparticles (NP). The so-called Tag-LIBS, i.e. a sensitive detection of specific biomarkers, was applied to detect the NP labels using the nanosecond ablation sampling. The NP labels were examined from the bottom of the standard 96-well microtiter plate. Thanks to the well-known flexibility in the LIBS instrumentation, the plasma emission collection as well as the focusing optics can be rebuilt into a collinear arrangement. The presented experiments proved the suitable detection of silver NPs from the microtiter plate’s bottom without any negative effects on the NPs’ qualitative and quantitative analysis. Subsequently, a sandwich immunoassay tha tdetects human serum albumin was successfully developed, using streptavidin-coated AgNP labels. The assay provided a detection limit of 10 ng/mL, which is comparable with the fluorescence-based readout. The main advantage of LIBS readout is that it enables a detection of any type of NP labels independent on their fluorescence or catalytic properties. Owing to the immediate signal response, a relatively simple instrumentation enabling the automation, and the capability of multi-elemental analyses, LIBS technique offers a promising and fast alternative to other readout techniques allowing a multiplex detection of various biomarkers.