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Integrating laser particles into live cells, tissue cultures, and small animals is an emerging and rapidly evolving technique that offers non-invasive interrogation and labelling with unprecedented information density. The bright and distinct spectra of laser particles make this approach particularly attractive for high-throughput applications requiring single-cell specificity, such as multiplexed cell tracking and intracellular biosensing. To be of practical relevance, the implementation of these applications requires high-resolution, high-speed spectral readout and advanced analysis routines, which leads to unique technical challenges. Here, we present a modular protocol consisting of two separate procedures. The first part of our protocol instructs users on how to efficiently integrate different types of laser particles into living cells. The second part presents a workflow for obtaining intracellular lasing spectra with high spectral resolution and up to 125 kHz readout rate and starts from the construction of a custom hyperspectral confocal microscope. We provide guidance on running hyperspectral imaging routines for various experimental design choices and recommend specific workflows for processing the resulting large data sets along with an open-source Python library of functions covering the entire analysis pipeline. The results one can obtain using this protocol are illustrated with three representative experiments: Rapid, large-volume mapping of absolute refractive index using polystyrene microbead laser particles, intracellular sensing to monitor cardiac contractility with polystyrene microbead laser particles, and long-term cell tracking using semiconductor nanodisk laser particles. Our sample preparation and imaging procedures can be completed within two days. Setting up the hyperspectral confocal microscope for laser particle characterization will take users with little prior experience in optical and software engineering <2 weeks to complete.

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