<p>Lineage tracing is a powerful tool to study cell history and cell dynamics during tissue development and homeostasis. An increasingly popular approach for lineage tracing is to generate high-frequent mutations at given genomic loci, which can serve as genetic barcodes to label different cell lineages. However, current lineage tracing mouse models suffer from low barcode diversity and limited single-cell lineage coverage. We recently developed the DARLIN mouse model by incorporating three barcoding arrays within defined genomic loci and combining Cas9 and terminal deoxynucleotidyl transferase (TdT) to improve editing diversity in each barcode array. We estimated that DARLIN generates 10¹⁸ distinct lineage barcodes in theory, and enables the recovery of lineage barcodes in over 70% of cells in single-cell assays. In addition, DARLIN can be induced with doxycycline to generate stable lineage barcodes across different tissues at a defined stage. Here we provide a step-by-step protocol on applying the DARLIN system for in vivo lineage tracing, including barcode induction, estimation of induction efficiency, barcode analysis with bulk and single-cell sequencing, and computational analysis. The execution time of this protocol is ~1 week for experimental data collection and ~1 d for running the computational analysis pipeline. To execute this protocol, one should be familiar with sequencing library generation and Linux operation. DARLIN opens the door to study the lineage relationships and the underlying molecular regulations across various tissues at physiological context.</p>