Process analytical technology has become a relevant topic in both industry and academia as a mechanism to control process quality by measuring critical parameters; being mainly applied in pharmaceutical industry. An emerging topic is process monitoring with subsequent process automation in flow chemistry using inline, online and atline analyzers. Flow chemistry often deliberately and favorably uses harsh conditions (termed Novel Process Windows) to achieve process intensification which raises the need for sampling under these conditions. This demands for setting in place a stabilization of the sample before exposing it to the processing. Ignoring this may result in being unable to use inline/online analytics and posing the need for a separation step before quantitative analysis, leaving atline analysis as the only feasible option. That means that sampling and connected operations need also to be automated. This is where this study sets in, and this is enabled by a modified high-performance liquid chromatography (HPLC) autosampler coupled to the photo-high-p,T flow synthesis of vitamin D₃. It shows that sampling variables, such as decompression speed, can be even more critical in terms of variability of results than process variables such as concentration, pressure, and temperature. The modification enabled the autosampler fully automated and unattended sampling from the reactor and enabled pressure independent measurements with 89% accuracy, >95% reproducibility, and >96% repeatability, stating decompression speed as the primary responsibility for measurements’ uncertainty.