Protein-RNA interactions are crucial for gene expression regulation, with their interface providing key structural insights into the underlying mechanisms. Photo-crosslinking can be applied to generate covalently connected peptide-RNA hybrids at the protein-RNA interaction interface. To date, the analysis of peptide-RNA hybrids by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been challenged by the inefficient isolation of these hybrids, low mass spectra quality, and complex data analysis. Recent studies have attempted to address these issues by chemically digesting the RNA moiety down to a single nucleotide, yet this approach compromises the sequence information of the RNA at the crosslink sites. This study reveals that non-crosslinked RNA is a major contaminant in the sample of photo-crosslinked protein-RNA hybrid, interfering in their LC-MS/MS detection in many ways. Here, we present a unifying workflow that efficiently removes free RNA from samples while preserving the crosslinked RNA, which can be tuned to the appropriate length for LC-MS/MS or single-molecule RNA sequencing. Using a novel high-intensity UV irradiation device, we achieved highly efficient protein-RNA crosslinking in living cells in their original culture medium within seconds of irradiation. Photo-crosslinked peptide-RNA hybrids were then isolated using a systematically optimized workup, which massively improved the LC-MS/MS identification and quantification of peptide-RNA hybrids. We showcase the performance of this method for tracking dynamic protein-RNA interactions upon drug treatment, recapitulating structural changes in known protein-RNA complexes. Overall, our study identifies confounding problems in the sample quality of peptide-RNA hybrids and presents an optimized workflow for their isolation that enables the analysis of protein-RNA interaction sites with a temporal resolution of seconds.