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Understanding cellular force transmission dynamics is crucial in mechanobiology. We developed the DNA-based ForceChrono probe to measure force magnitude, duration, and loading rates at the single-molecule level within living cells. The ForceChrono probe circumvents the limitations of in vitro single-molecule force spectroscopy by enabling direct measurements within the dynamic cellular environment. Our findings reveal integrin force loading rates of 0.5–2 pN/s and durations ranging from tens of seconds in nascent adhesions to approximately 100 s in mature focal adhesions. The probe's robust and reversible design allows for continuous monitoring of these dynamic changes as cells undergo morphological transformations. Additionally, by analyzing how mutations, deletions, or pharmacological interventions affect these parameters, we can deduce the functional roles of specific proteins or domains in cellular mechanotransduction. The ForceChrono probe provides detailed insights into the dynamics of mechanical forces, advancing our understanding of cellular mechanics and the molecular mechanisms of mechanotransduction. [Display omitted] • ForceChrono probe quantifies the "temporal codes" of mechanotransduction in cells • Reveals force duration from tens of seconds to 100 s in adhesion sites • Measures integrin force loading rates from 0.5 to 2 pN/s • ForceChrono probe reveals in-cell protein roles by measuring force dynamics Development of the ForceChrono probe allows for precise measurement of duration and loading rates of single-molecule forces in living cells, revealing protein roles in force dynamics. [ABSTRACT FROM AUTHOR]