A reliability study on anisotropically conductive adhesive joints on a Flip-Chip/FR-4 assembly has been carried out. In the study, nine types of anisotropic conductive adhesive (ACA) and one nonconductive film (NCF) were used. In total, nearly one-thousand single joints were subjected to reliability tests in terms of temperature cycling between −40°C and 125°C with a dwell time of 15 minutes and a ramp rate of 110°C/min. The test chip used for this extensive reliability test had a pitch of 100 μm. Therefore, this work was particularly focused on evaluation on the reliability of ultra fine pitch flip-chip interconnections using anisotropically conductive adhesives on a low-cost substrate. The reliability was characterized by single contact resistance measurement using the four-probe method during temperature cycling testing up to 3000 cycles. The Mean Time To Failure (MTTF) (defined as 50% failure of all tested joints) are 650, 2500, and 3500 cycles when the failure definition is defined as 20% increase, larger than 50 mΩ and larger than 100 mΩ, respectively, using the in-situ electrical resistance measurement technique. Using the discontinuous (manual) measurement at room temperature by taking out the sample from the cycling chamber, the MTTF for the same joint system is around 2500 cycles in the case that the failure criteria is defined as 20% of the resistance increase, far better than the results from the in-situ measurement. The results show clearly that in optimized conditions, high reliability flip-chip anisotropically conductive adhesive joints on low-cost substrate can be achieved.