Abstract Introduction A potential complication in critically ill patients is the formation of bone in soft tissues, termed heterotopic ossification. The exact pathogenetic mechanisms are still undetermined. Bone morphogenetic proteins induce bone formation, while signalling through the receptor activator of nuclear factor kappa-Β (RANK) and its ligand (RANKL), regulates osteoclast formation, activation, and survival in normal bone modelling and remodelling. Osteoprotegerin protects bone from excessive bone loss by blocking RANKL from binding to RANK. Aim The study aimed to investigate these molecules as potential prognostic biomarkers of heterotopic ossification development in critically ill patients. Materials and Methods In this prospective observational study, BMP-2, RANKL, and osteoprotegerin were measured by ELISA in twenty-eight critically-ill, initially non-septic patients, on admission to an ICU, seven days post-admission, and thirty days after ICU discharge. Results In the critically-ill cohort, nine of the twenty-eight patients developed heterotopic ossification up to the 30-day follow-up time-point. The patients who developed heterotopic ossification exhibited significantly reduced BMP-2 and RANKL levels on ICU admission, compared to patients who did not; Osteoprotegerin readings were similar in both groups. Conclusions Critically-ill patients who will subsequently develop heterotopic ossification, have significantly lower BMP-2 and RANKL levels at the time of ICU admission, suggesting that these proteins may be useful as prognostic markers for this debilitating condition.