The present research mainly focused on in vitro assay to measure intracellular activity and human recombinant α- glucocerebrosidase uptake. Human alpha glucocerebrosidae was family of lysosomal hydrolases, it used to enzyme replacement therapy and leads to deficiency of lysosomal storage diseases (LSD). Most people agree that using in vitro assays to assess a prospective compound's bioactivity before moving on to in vivo research is a helpful approach. Thorough assessments of a broad range of various substances can be performed with a specific focus on the anticipated bioactivity by utilising pertinent cell lines. In vitro studies three techniques were used for the quantification α-rhGC like flow cytometry extraction and activity assay and fluorescence microscopy was used to qualitatively evaluate the degraded intracellular substrate. The three techniques put into practice and put to the test proved to be useful instruments for examining the cellular uptake of rhGC. It was also possible to demonstrate the intracellular activity of a pertinent substrate using the fluorescence-based tests. The techniques are therefore very valuable for analysing future variations of enzymes expressed by P. pastoris strains that have been humanised.