The doctrine of the Holy Spirit is the most distinctive element of Assemblies of God theology. The AG affirms that the Holy Spirit is the third person of the Trinity, fully divine and coequal with the Father and Son. The Spirit convicts of sin, regenerates the believer, indwells every Christian, and distributes spiritual gifts. However, the AG goes further than most evangelical churches by teaching a distinct experience called 'the baptism in the Holy Spirit.'

AG theology holds that the baptism in the Holy Spirit is a second, definite work of grace subsequent to conversion, empowering the believer for Christian witness and ministry. Drawing on Acts 2 (Pentecost) and related passages, the AG teaches that the initial physical evidence of this Spirit baptism is speaking in other tongues (glossolalia) — a supernatural utterance in an unlearned language. This 'initial evidence' doctrine distinguishes the AG from other evangelical and charismatic groups.

Beyond tongues as initial evidence, the AG affirms the continuing operation of all spiritual gifts listed in 1 Corinthians 12, including prophecy, healing, miracles, and words of knowledge. These gifts are available to the church today and are exercised regularly in AG worship. The AG is explicitly non-cessationist, rejecting the view that miraculous gifts ceased after the apostolic era.