Although considerable progress has been made to optimize the optoelectronic properties of conjugated polymers (CPs), the rational design of CPs with tailored physical properties for end-use applications remains a significant challenge. Specifically, experimental characterization of conjugated polymer backbone conformations remains underexplored due to limited techniques that are capable of distinguishing the backbone and side-chain structures at nanoscopic resolution. Thus, relating the electronically functional backbone conformation to the material's macroscopic optoelectronic property is an ongoing challenge. Here, small-angle neutron scattering techniques (SANS) with contrast-variation (CV) experiments are employed on poly(3-alkylthiophenes) (P3ATs) with both deuterated and protonated side chains in a mixture of protonated and deuterated solvents to decouple the backbone and side-chain scattering signals. We obtained the form factor of P3ATs' backbone, side chains, and cross-scattering term by deconvoluting their respective scattering signals. Poly(3-decylthiophene) shows a persistence length of 1.05 +/- 0.1 nm for the conjugated polymer backbone and 2.10 +/- 0.2 nm for the entire chain. The strong scattering signal from long and flexible alkyl side chains leads to a seemingly more rigid conjugated polymer, which is further revealed by coarse-grained molecular dynamics (CG-MD) simulations. This work offers a methodology to decouple the scattering contribution from the CPs' backbone and side chains, thus elucidating the inherent conformation of the electronically active conjugated backbone, which provides guidance for the rational design of next-generation polymeric semiconductors.