At present, there is no reliable biomarker for the diagnosis of pancreatic ductal adenocarcinoma (PDAC). Due to this, together with the asymptomatic nature of the cancer, PDAC is often diagnosed at an advanced stage with an abysmal 5-year survival rate. Hence, a reliable biomarker for the timely diagnosis of PDAC is essential. The altered levels of protein expression are one of the hallmarks of cancer. Understanding these changes in PDAC could yield insights into the development of the disease and hence could potentially identify new biomarkers. Hence, in this study, mass spectrometry-based label‐free quantitative proteomics was employed to investigate the differently expressed proteins in the pancreatic cancer cell lines (Capan-2 and PANC-1), benign pancreatic cells (hTERT-HPNE) and normal pancreatic duct epithelial cells (H6c7). Furthermore, to study the circadian variations in the secretome of the pancreatic cell lines, the rhythmic expression of proteins was investigated at 6-hour intervals over 24 hours. The findings of this study provide novel insights into the processes and pathways involved in PDAC, potentially leading to the identification of circadian-based subtype-specific biomarkers.