Single-cell RNA sequencing (scRNA-seq) using metabolic RNA labeling enables detailed analysis of dynamic gene expression within single cells. However, most studies are limited to in vitro settings, restricting the exploration of in vivo transcriptomic dynamics. To address this, we developed scDyna-seq, a time-resolved scRNA-seq method for in vivo applications using 4-thiouridine (4sU) labeling. scDyna-seq efficiently captures nascent RNA, allowing for precise tracking of gene expression in both in vitro and in vivo contexts, including crossing the blood-brain and blood-fetal barriers. It is also compatible with other single-cell multiomics approaches. In a mouse bladder cancer model, scDyna-seq revealed that cisplatin (