Antinuclear antibodies (ANA) are significant biomarkers that are used to detect and classify autoimmune connective tissue disorders more efficiently. This paper provides a thorough overview of all the ANA profiling procedures, their testing advancements, clinical significance, and future directions. This study aimed to analyze the ANA profiles along with the prevalence of specific antibodies in patients presumed to have been diagnosed with autoimmune disorders in an Eastern Indian tertiary care hospital. The results were correlated with demographic data to conclude. ANA profiles of 48 patients from Peerless Hospitex Hospital, Kolkata were evaluated between January 24 to June 15, 2024. Out of them, 13 patients (27%) had positive ANA profiles. The most prevalent autoantibodies detected were SS-A and SS-B, which occurred in 7 patients (53.85%), followed by PM-Scl in 4 patients (30.77%). The study demonstrates how the ANA profile varies among different age groups, with the highest prevalence of 6 patients (46.15%) seen in the 60–75 age range. The clinical symptoms of diseases like autoimmune hepatitis, polymyositis, dermatomyositis, PSS (progressive systemic sclerosis), SLE (Systemic Lupus Erythematosus), and Sjogren's syndrome align with the ANA profile findings. This study offers insights into the distribution of autoantibodies targeting antigens present in ANA profiles, using clinical samples collected from a 550-bed tertiary care hospital in Eastern India. The findings underscore the need for comprehensive diagnostic approaches to autoimmune disorders.