DNA replication reactions are central to diverse cellular processes including development, cancer etiology, drug treatment, and resistance. Many proteins and pathways exist to ensure DNA replication fidelity and protection of stalled or damaged replication forks. Consistently, mutations in proteins involved in DNA replication are implicated in diverse diseases that include defects during embryonic development and immunity, accelerated aging, increased inflammation, blood disease, and cancer. Thus, tools for efficient quantitative analysis of protein interactions at active and stalled replication forks are key for advanced and accurate biological understanding. Here we describe a sensitive single-cell-level assay system for the quantitative analysis of protein interactions with nascent DNA. Specifically, we achieve robust in situ analysis of protein interactions at DNA replication forks (SIRF) using proximity ligation coupled with 5'-ethylene-2'-deoxyuridine click chemistry suitable for multiparameter analysis in heterogeneous cell populations. We provide validation data for sensitivity, accuracy, proximity, and quantitation. Using SIRF, we obtained new insight on the regulation of pathway choice by 53BP1 at transiently stalled replication forks.