<p>With the growing applications of gold nanoparticles (AuNPs) in healthcare and medical research, development of cost-effective, high-throughput methods for quantification of gold in AuNPs becomes highly desirable. The present work reports an optimized protocol for gold estimation utilizing simple reagent, ascorbic acid (AA) and termed as modified ascorbic acid (MAA) assay . Gold estimation by the in-house developed assay was validated by another sensitive technique of flame atomic absorption spectroscopy (FAAS), which showed significant correlation co-efficient value (<i>R</i>² = 0.98) in the range of 20–200&#xa0;ppm. Compared to other biologically relevant metal ions (e.g., Fe²⁺,Fe³⁺,Mg²⁺,Zn²⁺,Cu²⁺,Ca²⁺), the MAA assay showed specificity for gold and insignificant interference of most of other biologically relevant metal ions. To the best of our knowledge, no previous studies have demonstrated a method for in-situ bright-field microscopic visualization of AuNPs’ sub-cellular distribution and their quantification in human cells. MAA assay demonstrated sensitive visualization of AuNPs in cancer cells grown in multi-well plates as well as the estimation of AuNPs in treated cell pellets. The sub-cellular distribution of AuNPs determined by MAA in MCF-7 cells was validated by multi-photon microscopy and transmission electron microscopy. Thus, the present work provides a fast and robust method for gold estimation from AuNPs present in solutions or in biological samples with implications for enhancing the scope of AuNPs research.</p> Graphical Abstract <p></p>