Slides are transparent pieces of glass used to hold samples in biology and medicine. To improve real-time detection of their content in production environments, slides are ablated using a 1064-nm laser and the plasma's emission spectra are recorded for different experimental parameters. Herein, the Si I 288.16 nm, Ca I 422.67 nm, and Na I 589.00 nm lines are selected for analysis. The spectral intensity and signal stability are used as indices to optimize the key experimental parameters, i.e., spectral acquisition delay time, laser energy and repetition rate, ablation mode, and lens-to-slide distance. Results show that, for the experimental system of laser-induced slide ablation, the optimal spectral acquisition delay time is 3 μs and the optimal laser repetition rate is 2 Hz. A more stable spectral signal can be obtained using a single-point ablation method. In addition, for a lens with a focal length of 100 mm, the optimal focusing position is 8 mm below the slide surface; hence, the optimal lens-to-slide distance is 92 mm. The energy of each laser pulse is set to 150 mJ to avoid damaging the slide while still obtaining spectra with sufficiently highline intensities and signal stabilities to meet the experimental requirements. Optimizing the experimental system's key parameters in this manner provides an experimental basis for real-time, online, in-situ detection of slide using laser-induced breakdown spectroscopy.