Laser tweezers Raman spectroscopy (LTRS) is a variation of micro-Raman spectroscopy that is used to analyze single cells and biological particles suspended in an aqueous environment. The Raman spectrum of the cell/particle reflects its intrinsic biochemical composition and molecular structures. The technique utilizes a laser trap generated by a tightly focused Gaussian laser beam to physically manipulate individual cells and immobilize them in the laser focal volume. The same laser that is used for optical trapping also simultaneously excites Raman signals from the trapped cell, which are detected using a spectrometer and a confocal detection setup. LTRS offers unique capabilities not commonly found in other optical cytometry methods, such as label-free chemical analysis, multi-parametric chemical detection with a single excitation laser, and a non-photobleaching signal that can be used to quantitate and monitor dynamic chemical changes. This chapter provides guidelines on the design of a single beam LTRS microscope and methods for building and aligning the system. Operating procedures for trapping particles and acquiring spectra and a summary of data analysis techniques are provided.